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.     

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.     

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.     

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.     

Early development in the mouse: would it be affected by microgravity?

Gravity has been identified as a morphogenetic signal in Amphibian and bird embryonic development so it is plausible that it might be such in mammals as well. Since early mammalian development shows some apparently significant differences to these other groups, a brief summary of mouse embryogenesis will be given identifying events in which polarity is an important feature and consequently, in which gravity may be a causative factor. These include compaction and polarization during cleavage, establishment of the radial axis, the embryonic-abembryonic axis, the dorso-ventral axis, and the anterior-posterior axis, implantation, and the later rotation of the embryo. The experimental data on these morphogenetic steps will be discussed and an assessment of the possible involvement of gravity will be made.     

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.     

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.     

微重力下胶原蛋白纤维化及羟基磷灰石结晶的初步研究

在长期空间飞行过程中, 骨质丢失是一个严重问题. 羟基磷灰石(HAP)晶体是骨骼的主要成分, 骨骼中的胶原蛋白纤维在HAP生长结晶过程中起到关键作用. 研究了胶原蛋白纤维化过程在模拟微重力和常重力条件下的变化, 对以胶原 蛋白纤维作为模板生长出的HAP晶体形貌进行了观察. 结果表明, 不同浓度胶原蛋白溶液中形成的胶原蛋白纤维, 其内部孔隙数量和尺寸在模拟微重力条件下要明显大于常重力条件下, 胶原蛋白纤维内部孔隙的分布也不同于常重力条 件下的结果. 以模拟微重力条件下形成的胶原蛋白纤维为模板生长出的HAP 晶体主要为立方体状, 而以常重力条件下形成的胶原蛋白纤维为模板生长出的 HAP晶体形貌主要为板状. 该结果有助于未来进一步阐明空间骨质丢失的机理.      

Effect of microgravity on primordial germ cells (PGCs) in silk chicken offspring (Gallus gallus domesticus)

Primordial germ cells (PGCs), precursors of germline cells, display a variety of antigens during their migration to target gonads. Here, we used silk chicken offspring (Gallus gallus domesticus) embryos subjected to space microgravity to investigate the influence of microgravity on PGCs. The ShenZhou-3 unmanned spaceship carried nine fertilized silk chicken eggs, named the flight group, returned to Earth after 7 days space flight. And the control group has the same clan with the flight group. PGCs from flight and control group silk chicken offspring embryos were examined during migration by using two antibodies (2C9 and anti-SSEA-1), in combination with the horseradish peroxidase detection system, and using periodic acid-Schiff’s solution (PAS) reaction. After incubation for about 30 h, SSEA-1 and 2C9 positive cells were detected in the germinal crescent of flight and control group silk chicken offspring embryos. After incubation of eggs for 2–2.5 days, SSEA-1 and 2C9 positive cells were detected in embryonic blood vessels of flight and control group silk chicken offspring embryos. After incubation of eggs for 5.5 days, PGCs in the dorsal mesentery and gonad could also be identified in flight and control group silk chicken offspring embryos by using SSEA-1 and 2C9 antibodies. Based on location and PAS staining, these cells were identified as PGCs. Meanwhile, at the stage of PGCs migration and then becoming established in the germinal ridges, no difference in SSEA-1 or 2C9 staining was detected between female and male PGCs in flight and control group silk chicken offspring embryos. Although there were differences in the profiles of PGC concentration between male and female embryos during the special circulating stage, changing profile of PGCs concentration was similar in same sex between flight and control group offspring embryos. We concluded that there is little effect on PGCs in offspring embryos of microgravity-treated chicken and that PGC development appears to be normal.     

Features of vestibuloocular reflex modulations induced by altered gravitational forces in tadpoles (Xenopus laevis).

In Xenopus laevis tadpoles, we studied the static vestibuloocular reflex (rVOR) in relation to modifications of the gravitational environment to find basic mechanisms of how altered gravitational forces (AGF) affect this reflex. Animals were exposed to microgravity during space flight or hypergravity (3g) for 4 to 12 days. Basic observations were that (1)the development of the rVOR is significantly affected by altered gravitational conditions, (2) the duration of 1g-readaptation depends on the strength of the test stimulus, (3) microgravity induces malformations of the body which are related to the rVOR depression. Future studies are based on the hypotheses (1) that the vestibular nuclei play a key roll in the adaptation to AGF conditions, (2) that the stimulus transducing systems in the sense organ are affected by AGF conditions, and (3) that fertilized eggs will be converted to normal adults guided by physiological and morphological set points representing the genetic programs. Developmental retardation or acceleration, or otherwise occurring deviations from standard development during embryonic and postembryonic life will activate genes that direct the developmental processes towards normality.     

Possible actions of gravity on the cellular machinery.

Since the first flight of the ESA Biorack on the German Spacelab Mission D1 in 1985 evidence has been obtained that biological cells and small unicellular organisms function differently under conditions of microgravity. However, there is still lack of scientific proof that these effects are caused by a direct influence on the cells in the weightlessness condition. The question how normal gravity may play a role in cellular activity is being addressed and the results show that gravity may provide important signals during certain state transitions in the cell. These would be gravity-sensitive windows in the biological process. Also, by amplification mechanisms inside the cell, the cell may assume a state that is typical for normal gravity conditions and would change in microgravity. Experimental tools are discussed that would provide the conditions to obtain evidence for direct action of gravity and for the possible existence of gravity-sensitive windows.     

Regulative development of Xenopus laevis in microgravity.

To test whether gravity is required for normal amphibian development, Xenopus laevis females were induced to ovulate aboard the orbiting Space Shuttle. Eggs were fertilized in vitro, and although early embryonic stages showed some abnormalities, the embryos were able to regulate and produce nearly normal larvae. These results demonstrate for the first time that a vertebrate can ovulate in the virtual absence of gravity, and that the eggs can develop to a free-living stage.     

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.     

Evaluation of using two-phase frictional pressure drop correlations for normal gravity to microgravity and reduced gravity

The calculation of two-phase frictional pressure drop (TPFPD) is required by two-phase systems operating under microgravity and reduced gravity. There are a large number of correlations for the TPFPD in tubes under normal gravity. However, it is hard to find out a TPFPD correlation obtained from microgravity and/or reduced gravity conditions, and thus people have to use TPFPD correlations for normal gravity to calculate TPFPD under microgravity and reduced gravity. It is necessary to evaluate the feasibility of such practice. This paper offers a comprehensive review of the TPFPD correlations for normal gravity and an up-to-data survey of the TPFPD experimental study under microgravity and reduced gravity. There are 23 TPFPD correlations for normal gravity reviewed and 135 experimental data under microgravity obtained from the literature. These experimental data are used to evaluate the reviewed TPFPD correlations. It is found that the smallest mean absolute relative deviation (MARD) of the correlations is greater than 34%. Using TPFPD correlations for normal gravity to reduced gravity and microgravity may be acceptable for the first approximation, but correlations intended for microgravity and reduced gravity are needed and more experiments are desired to obtain more data with high accuracy.     

Vestibular and visual contribution to fish behavior under microgravity.

Vestibular and visual information are two major factors fish use for controlling their posture under 1 G conditions. Parabolic flight experiments were carried out to observe the fish behavior under microgravity for several different strains of Medaka fish (Oryzias latipes). There existed a clear strain-difference in the behavioral response of the fish under microgravity: Some strains looped, while other strains did not loop at all. However, even the latter strains looped under microgravity conditions when kept in complete darkness, suggesting the contribution of visual information to the posture control under microgravity. In the laboratory, eyesight (visual acuity) was checked for each strain, using a rotating striped-drum apparatus. The results also showed a strain-difference, which gave a clue to the different degree of adaptability to microgravity among different strains. Beside loopings, some fish exhibited rolling movement around their body axis. Tracing each fish during and between parabolas, it was shown that to which side each fish rolls was determined specifically to each individual fish, and not to each strain. Thus, rolling direction is not genetically determined. This may support the otolith asymmetry hypothesis. Fish of a mutant strain (ha strain, having homozygous recessive of one gene ha) have some malfunction in otolith-vestibular system, and their behavior showed they are not dependent on gravity. Morphological abnormalities of their ear vesicles during the embryonic and baby stages were noted. Their eyesight and dorsal light responses were also studied. Progress in the project of establishing a new strain which has good eyesight and, at the same time, being deficient in otolith-vestibular system was reported. Crosses between the strain of good eyesight and ha strain were made, and to some extent, F2 fish have already shown such characteristics suited for living under microgravity conditions.     

Influence of altered gravity on the cytochemical localization of cytochrome oxidase activity in central and peripheral gravisensory systems in developing cichlid fish.

Cichlid fish larvae were reared from hatching to active free swimming under different gravity conditions: natural environment, increased acceleration in a centrifuge, simulated weightlessness in a clinostat and near weightlessness during space flight. Cytochrome oxidase activity was analyzed semiquantitatively on the ultrastructural level as a marker of regional neuronal activity in a primary, vestibular brainstem nucleus and in gravity receptive epithelia in the inner ear. Our results show, that gravity seems to be positively correlated with cytochrome oxidase activity in the magnocellular nucleus of developing fish brain. In the inner ear the energy metabolism is decreased under microgravity concerning utricle but not saccule. Hypergravity has no effect on cytochrome oxidase activity in sensory inner ear epithelia.     

重力对单气泡生长特性的影响

采用VOF (Volume of Fluid)多相流模型, 通过用户自定义函数UDF (User Defined Function)实现相变过程中质量和能量的输运, 对微重力条件下尺寸为10mm × 10mm × 25mm的矩形通道的池沸腾现象进行数值模拟, 得到了微重力及常重力作用下单个气泡生长特性的差异. 模拟结果表明, 微重力条件下气泡周围的流线与温度场的分布有显著差异; 由于表面张力作用, 微重力下的气泡脱离特性与常重力下不同; 在微重力条件下, 气泡直径的变化比较复杂, 并与重力加速度的大小有关; Marangoni流对微重力下的流动影响很大, 使换热系数波动, 而且波动的幅度随重力加速度的减小而增大.      

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.