Effects of a long-term spaceflight on immunoglobulin heavy chains of the urodele amphibian Pleurodeles waltl.

A variety of immune parameters are modified during and after a spaceflight. The effects of spaceflights on cellular immunity are well documented; however, little is known about the effects of these flights on humoral immunity. During the Genesis space experiment, two adult Pleurodeles waltl (urodele amphibian) stayed 5 mo onboard Mir and were subjected to oral immunization. Animals were killed 10 days after their return to earth. IgM and IgY heavy-chain transcripts in their spleens were quantified by Northern blotting. The use of the different VH families (coding for antibody heavy-chain variable domains) in IgM heavy chain transcripts was also analyzed. Results were compared with those obtained with ground control animals and animals reared in classical conditions in our animal facilities. We observed that, 10 days after the return on earth, the level of IgM heavy-chain transcription was normal but the level of IgY heavy-chain transcription was at least three times higher than in control animals. We also observed that the use of the different VH families in IgM heavy-chain transcripts was modified by the flight. These data suggest that the spaceflight affected the antibody response against the antigens contained in the food.

Mouse bone marrow contains large numbers of functionally competent neutrophils.

The mouse has become an important model for immunological studies including innate immunity. Creating transgenic mice offers unique possibilities to study gene-function relationships. However, relatively little is known about the physiology of neutrophils from wild-type mice. Do they behave like human neutrophils, or are there species-specific differences that need to be considered when extrapolating results from mice to humans? How do we isolate neutrophils from mice? For practical reasons, many studies on mouse neutrophils are done with bone marrow cells. However, human bone marrow neutrophils appear to be heterogeneous and functionally immature. We have isolated and compared neutrophils from mouse bone marrow and from peripheral blood obtained by tail bleeding. Using the same Percoll density gradient for both preparations, we have obtained morphologically mature neutrophils from bone marrow and blood. Both cell populations responded to formylmethionyl-leucyl-phenylalanine (fMLF) with primary and secondary granule release and superoxide production. Quantitative analysis of our data revealed minor differences between cells from bone marrow and blood. Superoxide production and primary granule release were stimulated at lower fMLF concentrations in blood neutrophils. However, the amplitude and the kinetics of maximal responses were similar. The principal difference was the lifespan of the two cell populations. Bone marrow cells survived significantly longer in culture, which may suggest that they are receiving antiapoptic signals that are absent in the blood. Our data suggest that mice have a large reservoir of functionally competent neutrophils in their bone marrow. This reservoir may be needed to replace circulating neutrophils rapidly during infection.

Developmental biology of urodele amphibians in microgravity conditions.

Among the urodele amphibians, only Cynops pyrrhogaster and Pleurodeles waltl, two species of the Salamandridae family, were used in space experiments. The advantages for using urodeles reside (i) in reproduction: a few months after natural breeding, females can lay eggs in absence of males after a hormonal treatment, because spermatozoa were preserved in the cloacal pelvic glands of matted females, (ii) in the rate of development which is slower in Cynops and Pleurodeles than in the anuran Xenopus, (iii) in their physiological properties: they can live in a closed water container or in a moisturized environment, and they can fast during several days. Moreover, urodeles have an important phylogenetic interest. Many biological phenomena differ from those of anurans, such as fertilization events, the germ cell origin and the migration toward the differentiating gonads, and their regeneration capabilities. The main goals of the space experiments were to answer the following questions. On the one hand, does fertilization occur normally in microgravity? Is subsequent embryonic development normal in microgravity? Is further development and reproduction normal after return to Earth? On the other hand, does microgravity affect the organs in adult animals? Does microgravity affect the regeneration of organs? Fertilization in space is clearly demonstrated. However, subsequent embryonic development appears to be altered in microgravity. In Pleurodeles, abnormalities such as cortical cytoplasmic movements, decrease of cell adhesion, and loss of cells were observed. Although, early development was not strictly normal as a consequence of embryological regulation phenomena, young hatching larvae had normal morphological phenotypes and swimming behavior. After landing, no differences were observed between born-in-space animals to standard ones during the embryonic development to adulthood. The analyses of their offspring demonstrated that the percentages of fertilization and development were in accordance with the control animals. No genetic abnormalities were detected during the analysis of the offspring. The development of their progenies were also without characteristic differences compared to control Pleurodeles. Microgravity seems to have effects on the morphological and histological structures of organs of flight adults. However, as was the case in several experiments the number of analyzed adults was low, and it is too early to conclude on specific effects of microgravity. Moreover, in certain flights the temperature was not regulated, and an increase in temperature occurred. Conditions of these space flights had certainly influenced the samples, and consequently the interpretations of results. Space flights have clear effects on organs in regeneration. But more specifically, they have long term effects that last several weeks after the return of the animals to Earth. A similar result was also obtained for otoconia several months after landing. So far, however, no clear hypothesis could be proposed to interpret these observations.

Effects of space environment on embryonic growth up to hatching of salamander eggs fertilized and developed during orbital flights.

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.

An alternative internal splicing site defines new Ikaros isoforms in Pleurodeles waltl.

The Ikaros gene encodes a family of transcription factors which plays a crucial role in hematopoiesis. To improve our knowledge about the immune system of Pleurodeles waltl, we sequenced the cDNA coding for the Ik-1 isoform of that salamander and analyzed its tissue expression by semi-quantitative RT-PCR. Ikaros transcripts are abundant in the thymus and the spleen, thereby confirming that these organs are, respectively, the primary and secondary lymphoid tissues of Pleurodeles. Analysis of the isoforms produced by this animal revealed two isoforms characteristic of amphibians in which an alternative internal splicing site deletes the 3' half of exon 3 which interestingly comprises the first Zn finger of Ikaros. Ikaros transcripts were found at the earliest stages of development of Pleurodeles indicating that Ikaros has a function at the very early lymphopoietic stages. Moreover, the presence of Ikaros transcripts in spermatozoa suggests that this protein could have another and yet unknown function.

Sequencing and expression of the CD3 gamma/delta mRNA in Pleurodeles waltl (urodele amphibian).

The CD3 complex is an essential component of the T-cell receptor (TCR) implicated in T-cell maturation and activation. This TCR has been identified in both cartilaginous and bony vertebrates. In different studies where the CD3 chains were cloned and sequenced, it appeared that the CD3 complex is composed of several chains, all susceptible to phosphorylation and able to transduce signals. Here, by an approach combining degenerative oligonucleotide primers and RACE-PCR, we report the cloning and sequencing of a CD3 cDNA from the salamander Pleurodeles waltl, highly homologous to the Xenopus and chicken CD3 gamma/delta cDNAs. Using semi-quantitative PCR and Northern blot analysis, we found the highest CD3 gamma/delta mRNA expression in the thymus; weaker expression was observed in the spleen and blood, followed by the intestine, therefore confirming the tissue and lymphoid specificities of this mRNA. The signals in the spleen, blood and intestine represented 55%, 33% and 16%, respectively, of the signal detected in the thymus. During the embryonic and larval stages of Pleurodeles waltl development, CD3 gamma/delta mRNA expression begins early at the neurula stage (stage 15, 69 h after laying), increases up to stage 33 (9 days after laying) and afterwards remains stable, at least until the larval stage 42 (28 days after laying). As the thymus primordium appears much later, the question of the formation and maturation of the first T-cell precursors outside this organ is posed.

An European pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS.

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.

Melanin as a natural germ cell marker for intraspecific transplantation experiments in Ambystoma mexicanum (Urodela, Amphibia).

In urodele amphibians, the lack of a reliable germ cell marker restricts the experimental study of the germ lineage. In the present work, we conducted genetic and histological analyses in order to demonstrate that melanin from oocytes constitutes a germ cell marker available for intraspecific experiments in Ambystoma mexicanum. Then, using this marker, we implanted germ cells from undifferentiated gonads (stage 48) into the blastocoel of host embryos and investigated their fate and determined state. Our results show that, from this stage on, the donor cells do not differentiate into other cell types; therefore, they are restricted in developmental capacity and irreversibly determined as germ cells. On the other hand, exogenous germ cells were found in an isotopic position until the young tail-bud stage, and then were found in an ectopic position; these results suggest that, from the middle tail-bud stage on, an active process contributes to migration of primordial germ cells to the gonadal territory.

[Effect of temperature, ageing and hormonal conditions (thyroxine) on cell proliferation, in the young tadpole and during metamorphosis of the toadBufo bufo L. (Amphibia, Anura)]. / Influence de la température, de l'âge et des conditions hormonales (thyroxine) sur la prolifération cellulaire chez la jeune larve et pendant la métamorphose du crapaudBufo bufo L. (Amphibien Anoure).

Generation time, duration of cell cycle phases and growth fraction were measured for three tissues: telencephalon, anterior limb-bud mesenchyme and intestinal epithelium.By rearing young larvae at different temperatures (12, 16 and 24° C), it was demonstrated that: cell cycle duration (T) and the duration of S, G2 and M phases are shorter at higher temperature; the G1 phase is characteristically lengthened or shortened, depending on the tissue concerned at a given stage, the growth fraction (CP) is characteristic for each tissue, and does not vary with temperature the sensitivity to temperature conditions is also characteristic for each tissue; anterior limb bud mesenchyme is the least sensitive. Ageing and differentiation of cell populations during larval life and natural metamorphosis (at the constant temperature of 16° C) lengthen the generation time (T) and the duration of S, M and G1 phases; simultaneously the growth fraction decreases. But at the metamorphic climax, the growth rate of some tissues (telencephalon, secondary intestinal epithelium) is suddenly and temporarily increased, as a result of both a shortening of T and an increase of CP. On the contrary, other tissues (primary intestinal epithelium) no longer proliferate, and collapse. These different modifications to the different organs seem to be related to variations in the amount of thyroxine in the circulating blood. They can be considered as one of the aspects of the differential tissue sensitivity to thyroxine.