In lower vertebrates, gravity deprivation by orbital flights modifies the vestibuloocular reflex. Using the amphibian Xenopus laevis, the experiments should clarify to which extent macular structures of the labyrinth are responsible for these modifications. In particular, the shape of otoconia and number and size of sensory macular cells expressing CalBindin were considered. CalBindin is common in mature sensory cells including vestibular hair cells and is probably involved in otoconia formation. Two developmental stages were used for this study: stage 26/27 embryos, which were unable to perform the roll-induced vestibuloocular reflex (rVOR) at onset of microgravity, and stage 45 tadpoles, which had already developed the reflex. The main observations were that the developmental progress of the animals was not affected by microgravity; that in the young tadpole group with normal body shape the rVOR was not modified by microgravity, while in the older group with microgravity experience, the rVOR was augmented; and that significant effects on the shape of otoconia and on the number and size of CalBindin-expressing cells of the labyrinthine maculae cells were absent. In addition, behavioural data were never significantly correlated with morphological features of macular structures such as size and number of CalBindin-expressing cells. It is postulated that mechanisms of vestibular adaptation to microgravity during early development are probably based on mechanisms located in central structures of the vestibular system.

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.

In vertebrates, only few experiments have been performed in microgravity to study the embryonic development from fertilization. To date, these concern only amphibian and fish. We report here a study on the embryonic development of Pleurodeles waltl (urodele amphibian) eggs oviposited in microgravity. The experiment was performed twice on board the Mir space station and the data obtained included video recording and morphological, histological and immunocytological analyses. The data confirm that the microgravity conditions have effects during the embryonic period, particularly during cleavage and neurulation, inducing irregular segmentation and abnormal closure of the neural tube. Moreover, we observed several abnormalities hither to undescribed corresponding to cortical cytoplasm movements, a decrease of cell adhesion and a loss of cells. These abnormalities were temporary and subsequently reversible. The young larvae that hatched during the flight displayed normal morphology and swimming behavior after landing. The results obtained in the urodele Pleurodeles waltl are in accordance with those observed earlier in the anuran Xenopus laevis and in the fish Oryzias latipes.

Viable chimeras were constituted with two cranial and caudal complementary pieces of embryos derived from two distinct histocompatible AA and BB strains, which were incompatible with each other. The embryonic gonads of the resulting chimeras constituted two homo‐ or heterosexual territories. In most heterosexual chimeras, the testicular territory sex reversed the ovarian territory. The offspring analysis of a male chimera conclusively proved that ZW germ cells derived from the posterior female piece differentiated into spermatozoa. Nevertheless, the opposite situation was also demonstrated with a female chimera in which ZZ germ cells derived from the anterior male piece differentiated into oocytes. These gametogenesis reversions were tested by genetic and immunogenetic analyses of chimera offspring. The phenomenon of tolerance or rejection of skin allo‐ and autograft was used as a marker of origin of the chimera germ cells, which had produced the offspring. Moreover, in the first stage of the study, the origin of the pieces of adult chimeras was determined using skin grafts. During this stage, the embryonic tolerance was confirmed by the acquisition of four pieces of pairs of chimeras, and by the preservation of skin immunogenicity that was derived from each piece of the chimeras.

The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl . The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground‐born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.

Effects of microgravity (microG) on fertilization were studied in the urodele amphibian Pleurodeles waltl on board the MIR space station. Genetic and cytomorphologic analyses ruled out parthenogenesis or gynogenesis and proved that fertilization did occur in microG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in microG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the microG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in microG and in 1.4G, 2G, and 3G hypergravity showed for the first time that, compared to eggs in 1G, several characteristics of the fertilization process including the cortical reaction and the microvillus transformations were altered depending on the gravitational force applied to the eggs. Microvillus elevation, the most evident feature, was reduced on microG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of microG-eggs were observed.

A research group in our laboratories works on development of amphibians under microgravity conditions. Several experiments were performed in space conditions using embryos or adults of the urodele amphibian, Pleurodeles waltl ( Bautz et al ., 1995 ; Dournon et al., 1997; Husson et al., 1998; Duprat et al., 1998; Aimar et al., 2000 ). In 1999, for the so-called Perseus French space mission onboard the MIR space station, the project was to rear embryos and larvae of Pleurodeles waltl in microgravity conditions to study the appearance and evolution of otoconia in the inner ear (Oukda et al., 1999a and b). The present paper reports the technique used to feed and rear Pleurodeles larvae in microgravity conditions with the assistance of a cosmonaut.

The aim of this study is to determine the stages of appearance, morphology, crystallographic structure and chemical composition of otoconia during the inner ear development of an urodele amphibian, Pleurodeles waltl. The first otoconia are detected in the otocyst. Near hatching, calcitic otoconia are polyhedral in the saccule and cylindrical in the utricle. During the following stages, the saccular otoconia agglomerate and constitute a polyhedral calcitic otolith. At larval stage 44, aragonitic fusiform otoconia appear on the otolithic surface. At stage 52, X-ray diffraction analysis shows calcite and aragonite patterns. In adults, all the saccular otoconia are aragonitic. In contrast, the utricular otoconia do not show any modification up to adulthood. In the endolymphatic sac, otoconia appear at stage 45 and in the lagena at stage 49. They remain aragonitic up to adulthood. Energy dispersive X-ray spectroscopy (EDXS) elemental analysis of the otoconia reveals a high quantity of calcium with trace quantities of sodium, magnesium, phosphorus, sulfur, chlorine and potassium. However, magnesium and sulfur have a lower concentration in lagenar aragonitic otoconia than in utricular and saccular calcitic ones. As in adults, trace amounts of strontium are only found in aragonitic otoconia.

The aim of the present study was to define the morphology and the crystallographic and chemical composition of otoconia in different regions of the inner ear in Pleurodeles waltl (urodele amphibian). The inner ear of adults was microdissected and otoconia were analyzed by scanning electron microscopy (SEM), X-ray diffraction, energy dispersive X-ray (EDX) and transmission electron microscopy. Two types of crystals were detected by SEM. Otoconia had different shapes depending on their location in the membranous labyrinth. One type had a cylindrical body with a triplanar smooth facet at each end, the other ones had either a prismatic shape with flat sides and end faces or a fusiform shape with rounded body and pointed end. The forms corresponded to those previously identified by other authors. These two types of otoconia had different X-ray diffraction patterns. The cylindrical otoconia were calcitic and located in the utricle, the other ones were aragonitic and located in the saccule, lagena and endolymphatic sac. An analysis by EDX indicated that both types of otoconia contained about 95% calcium with trace quantities of sodium, magnesium, phosphorus, sulfur, chlorine and potassium. Trace amounts of strontium was only found in the aragonitic otoconia.

Anomalie m.p. is a spontaneous and heritable hindlimb abnormality described earlier. Twenty years later, Pleurodeles waltl larvae from the strain bearing anomalie m.p. and reared at room temperature or at 30°C, expressed abnormalities (ectrodactylia, hemimelia, ectromelia). A morphological study of all the hindlimbs and an analysis of the hindlimb skeleton of samples from the experimental animals confirmed that most of the skeletal malformations were identical to those previously reported and affected the disto‐proximal and prepostaxial pattern of the hindlimb. Analysis of the effects of rearing temperature on the expression of anomalie m.p. showed that the effects varied according to the developmental period at which the heat treatment was applied; the sooner the heat treatment began, the more numerous and more various were the degrees of severity of the malformations. Moreover, heat treatment induced the expression of two additional malformations not yet described: the first one, named ‘reversed knee joint’, was characterized by a reversal of the knee joint, and the second one, named ‘twisted foot’, by a downward twisting of the foot. The epigenetic effects of rearing at 30°C on hindlimb development are discussed with regard to the differentiation or patterning.

The modification of peristaltic activity in the presence of several metal ions has been investigated in the rat intestine by the isolated organ technique. The metals tested modify the intestinal movements: aluminum, chromium, and yttrium cause a decrease of amplitude, while iron showed no effect. By use of microscopic techniques, the presence of yttrium hydroxide was observed in the intestinal tissues. Iron also appears as a precipitate outside of the intestinal serosal, which may explain why iron did not modify the peristaltism. Chromium and aluminum were not apparent to microscope, despite being detected and quantified in the tissues by means of atomic emission spectrometer. We conclude that the trivalent ions of these elements may operate differently on the mechanisms of intestinal contractions: yttrium precipitates in intercellular spaces, iron precipitates outside the intestines, and chromium and aluminum remain in solution and are distributed homogeneously in the smooth intestinal muscle.

The role of aluminum accumulation in articular tissues of patients affected by dialysis-associated arthropathy (DAA) is questioned. The aim of this work is to identify the nature of these aluminum accumulations by the use of secondary ion mass spectrometry (SIMS). Al/Si ratios of about 1, measured by SIMS, strongly suggest for the first time the presence of aluminum silicates and possibly aluminum hydroxides in amyloid synovial tissue and articular cartilage of 1 patient with DAA and aluminum intoxication. This is thermodynamically consistent with the total dissolved Al and Si contents and pH measured in the synovial fluids. These results are similar to the abnormal Al distribution recently found by SIMS in the forebrain of chronic renal dialysis patients and to the amorphous aluminum silicates identified in the core of senile plaques in Alzheimer's disease.

The existence of factors mediating dithiothreitol (DTT) activation of sorghum (C4 plant) leaf NADP malate dehydrogenase has been shown. However, it seemed that the enzyme can be directly activated by the dithiol but with a lower magnitude. A treatment with pronase established the proteic nature of the factors. By chromatography on a DEAE cellulose column, at least three peaks of activity were identified. This elution profile was different from that obtained in the case of a C3 plant (French bean). Nevertheless, a cross experiment between enzymes and factors from the two types of plants indicated that they are interchangeable with regard to the activation process.