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a Laboratoire d'Immunologie Comparée, Université P. et M. Curie, 75253 Paris cedex 05, France
b EA 2401 Génétique et Interactions Cellulaires en Reproduction, Université Henri Poincaré, 54506 Vandoeuvre-lès-Nancy cedex, France
c Centre de Biologie du Développement, UMR 5547 CNRS, Université Paul Sabatier, Toulouse, France
ABSTRACT
Effects of microgravity (µG) 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 µG. Actual fertilization was demonstrated by the analysis of the distribution of peptidase-1 genes, a polymorphic sex-linked enzyme, in progenies obtained in µG. Further evidence of fertilization was provided by the presence of spermatozoa in the perivitelline space and in the fertilization layer of the µG eggs and by the presence of a female pronucleus and male pronuclei in the egg cytoplasm. Experiments in µG 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 µG-eggs and amplified on eggs submitted to 2G and 3G. No lethal consequences of these alterations on the early development of µG-eggs were observed.
developmental biology, fertilization
INTRODUCTION
Up to now, the occurrence of an actual fertilization process under microgravity (µG) conditions has not been clearly demonstrated in amphibians. Most experiments concerning development in µG resulted from natural fertilization previously performed on the ground or of artificial fertilization conducted in µG [1–7]. Actual artificial fertilization and various stages of embryonic development in µG were obtained on board sounding rockets and American shuttles using the anuran amphibians Rana pipiens and Xenopus laevis, the fertilization of which is external and monospermatic [5–7]. Some Xenopus embryos seemed to bypass developmental abnormalities and differentiated into quite normal larvae [6, 8]. Concerning urodele amphibians, characterized by internal and polyspermatic fertilization, the results of an experiment dedicated to the fertilization and development in µG of Cynops pyrrhogaster eggs have still not been published [9]. In another phylum, eggs of Oryzias latipes fertilized in µG developed successfully [10]. In the absence of genetic markers, the inheritance patterns of all the embryos that developed in µG are not known and the cytological features of the fertilization process in µG have not been fully investigated.
To ascertain that the development of amphibian eggs obtained under µG conditions was the consequence of true fertilization and not of parthenogenesis or gynogenesis, fertilization in space of Pleurodeles waltl (a urodele amphibian) was investigated. Embryos developed from eggs fertilized in µG were tested for their genetic inheritance patterns. Furthermore, cytological and morphological investigations were conducted to define the cellular events of the fertilization processes in µG up to the first cleavage. Moreover, we paid special attention to the microvilli (MV) of the egg surface that were compared in eggs submitted from µG to 3G gravitational forces. The results provided evidence that for P. waltl, true fertilization occurs in µG. Microgravity and hypergravity altered some morphological characteristics of fertilization with modifications of the pattern and the reorganization of the cortical pigmentation at fertilization. As a consequence, at the subcellular level, the molecular structures of the eggs were modified, suggesting an effect of gravity on cytoskeletal components. All transformations occurred without lethal consequences for later development.
MATERIALS AND METHODS
Strategy Used for the Space Experiments
Two replicate experiments, named FERTILE, were conducted on board the MIR space station during the French missions "Cassiopeia" and "Pegasus" synchronously to control experiments that were performed in a laboratory on the ground.
Before the space mission, P. waltl females were inseminated on the ground, 1–4 mo before each of the space experiments, and then reared at 8°C up to the launch time. The eggs from these natural fertilizations were examined to select 12 females for each of the space experiment (6 on MIR, 6 on the ground). The female newts kept spermatozoa in their cloaca ready to fertilize eggs after hormonal stimulation of ovulation [11].
During the space mission, a second ovulation and egg laying were provoked by a hormonal injection of the inseminated females with 1.5 µg LHRH (Sigma, St. Louis, MO). Under these conditions, as after natural mating, egg laying occurred with a 24- to 48-h delay after the hormone stimulation. The eggs were distributed into twin batches of 20 eggs and reared at 18–20°C. The first ones were stored in ambient gravity, and the other ones were placed in a centrifuge. Eggs were maintained alive or fixed with a 2% formaldehyde solution at 2-h intervals from egg laying (To time) up to the first cleavage, i.e., 6 h postfertilization, according to the table of normal development for P. waltl [12].
The space missions were 16 and 22 days long, respectively. Aboard MIR, the female newts were in µG conditions for 4 and 9 days before egg laying, respectively. Thus, the embryos remained 12 and 13 days in µG. Ambient gravity was µG in MIR and 1G in the laboratory. Consequently, centrifuges, running at the same speed (1G) aboard MIR and on the ground, provided 1G aboard MIR and 1.4G as a result of the centrifugal and gravitational forces in the control devices on ground. Aboard MIR, at each time point of access to the centrifuge, the egg samples were submitted to µG for 3 min. Fixed and living embryos developed in µG and in 1G aboard the space station and in 1G and 1.4G on ground were available for further analysis. In addition, eggs were centrifuged at 2G and 3G in the ground laboratory for 2, 4, and 6 h after laying. For clarity, eggs of these subsequent batches were referred as µG-, aboard 1G-, ground-1G-, 1.4G-, 2G-, and 3G-eggs, respectively. The ground control animals and eggs were treated identically to the animals and eggs on board MIR except for gravity as described. All animals were treated according to the principles expressed in the 1983 declaration of Helsinki.
After the space mission, the number of spermatozoa, the MV, and the morphology of fixed eggs and embryos were analyzed. Living embryos were observed during their development and used for the peptidase test.
Embryos and Larvae Developed Aboard MIR and in the Ground Laboratory
The 12 aboard females provided 327 µG-eggs and 89 1G-eggs for fixation before the two-cell stage aboard MIR and embryos and young larvae for development in µG aboard MIR. The percentage of development calculated for the two space missions was 56%. This percentage was defined as the number of living embryos at the two-cell stage or later stages from the number of the eggs in the spawning. After the landing, 51 larvae were kept alive and reared in the laboratory.
The 12 ground control females provided 203 1G-eggs and 61 1.4G-eggs that were fixed before the two-cell stage. These eggs were analyzed in comparison to those laid aboard MIR. Moreover, embryos and young larvae developed in 1G ground control devices. For these animals, the percentage of development was 51% for the two space missions. Sixty-six embryos were kept alive after the landing time and reared in the laboratory.
Peptidase-1 Test
In order to ascertain the genotype filiations, parents and their larvae born during the space flights were submitted to the peptidase-1 test. This test was based on the detection of peptidase activity. Erythrocytes or tail tip fragments from adults or larvae were homogenized as previously described [13]. The homogenates were analyzed by electrophoresis on starch gels followed by peptidase-1 activity detection according to Lewis and Harris [14] modified by Nicholson and Kim [15]. Peptidase-1 hydrolyzed specifically the dipeptide valyl-leucine that was used as substrate for the detection of enzyme activity based on the following reactions:
Peptidase-1 is a sex marker that allows identification of the different sex genotypes of P. waltl. Two codominant genes encode this dimeric and polymorphic enzyme: pep-1A carried by the sex chromosome Z, and pep-1B or its allele pep-1ß carried by the sex chromosome W. Males have a ZAZA genotype and females a ZAWB or a ZAWß genotype. However, in P. waltl, females selected by a particular cross have a WBWB or a WßWß genotype [16]. Each genotype gives a specific electrophoretic pattern on starch gels. The ZAZA males are characterized by one slowly migrating homodimeric band (aa), and the ZAWB or the ZAWß females by three bands: a BB or ßß fast homodimeric band, an AB or Aß medium heterodimeric band, and an AA slow homodimeric band. The WBWB or the WßWß females are characterized by one BB or ßß fast homodimeric band [16, 17].
Spermatozoa Counting
Presence of spermatozoa in batches of 15 eggs fixed 2 h after laying was detected using phase contrast or fluorescent microscopy after staining in 4 mg 4',6-diamidino-2-phenylindole in 10 ml physiological medium solution (Boehringer GmbH, Mannheim, Germany). In 2-h eggs, the male nuclei of the spermatozoa became pronuclei. On the egg surface, the cortical reaction was affirmed by the migration of the cortical pigment toward the animal pole and by the disappearance of the maturation spot at the animal pole. Only the spermatozoa located in the perivitelline space or inserted in the fertilization layer were counted, excluding spermatozoa located in the jelly coat.
Cytology
Cytological and ultrastructural examinations of the eggs were done by applying standard protocols for light and transmission or scanning electron microscopy (TEM or SEM). In the latter case, the electron micrographs were scanned and monitored with a computer equipped with NIH image analysis software. The analyzed surface was limited to 50 µm x 50 µm of the region surrounding the animal pole. Diameter, height, and density of MV and the influence zone area were statistically analyzed by calculation of mean and standard deviation, and Students t-test (0.01 level). The influence zone area, which is defined by mathematical analysis, allowed quantification of the distribution of MV on the egg surface and measured the mean distances between neighboring MV. The results were expressed in percentage of surface units of influence zone area, here <2 µm2, 2–4 µm2, and >4 µm2.
RESULTS
Activated Eggs after Hormonal Stimulation
The percentage of activated eggs obtained after hormonal stimulation and expressing a cortical reaction, i.e., the migration of the cortical pigment toward the animal pole and the disappearance of the maturation spot at the animal pole, was calculated from samples of eggs, 188 of 327 µG-eggs and 102 of 203 ground control 1G-eggs. For the first mission, the percentage of activated eggs was 75% and 49% for the six aboard females and six ground control females, respectively. For the second mission, the percentage of activated eggs was 91% and 90% for the six aboard females and six ground control females, respectively. In addition, the percentage of egg development was 21% and 82% for the µG aboard females, and 23% and 78% for the 1G ground control females, for the first and second mission, respectively (Table 1). These data reflected the fact that the first space mission occurred out of the natural breeding season. However, the good and comparable yield of activated eggs aboard MIR and in the ground laboratory indicated that the weightless conditions were appropriate for fertilization.
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Urodele Eggs Are Fertilized in µG
Presence of spermatozoa in the perivitelline space and fertilization layer and of male and female pronuclei in the egg cytoplasm The presence of spermatozoa in the perivitelline space and of spermatic spots on the surface of some µG-eggs was an indication of the occurrence of fertilization. Spermatozoa were observed on eggs laid in weightless conditions, and some of these eggs exhibited typical spermatic impacts on their surface (Table 1). For the first mission, differences were observed between the number of spermatozoa associated with eggs resulting from natural fertilization (mean number = 6, range 0 to 10), eggs obtained aboard MIR (mean number = 4, range 1 to 14), and the ground control eggs (mean number = 1, range 0 to 2). These differences were in accordance with the decreasing percentages of activated eggs: 90% for natural spawning, 75% for aboard eggs, and 49% for ground controls. They seemed unrelated to weightlessness, because for the second mission eggs laid by the females selected for MIR and then eggs laid by these females aboard MIR displayed the same number of spermatozoa and the same percentage of activation as the ground control eggs, respectively (Table 1). Moreover, the mean number and range of spermatozoa were higher in eggs laid by the females selected for MIR than in eggs from ground control females. These data were consistent with the criteria of selection of females for the space flights. The females were selected after a natural mating depending on the number of spermatozoa in the perivitelline spaces and fertilization layers of their eggs (Table 1).
As a rule, in P. waltl, 1 to about 14 spermatozoa enter the oocyte, but only one male pronucleus fuses with the female pronucleus [18, 19]. Each of the 10 examined µG-eggs and control eggs included one female pronucleus, located on the animal-vegetal axis, and 2 to 12 male pronuclei, characterized at this stage by their lateral position and their smaller size in comparison with the female pronucleus (Fig. 1). These pronuclei appeared quite normal.
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Sex genotype of the larvae developed in µG In P. waltl species, all the spermatozoa of the ZAZA genetic males carry the peptidase-1 gene A. The gametes of ZAWß/B genetic females display the peptidase-1 gene A, or the peptidase gene ß, or B. If parthenogenesis or gynogenesis occurred during weightlessness, the genotype of the resulting animals would be WßWß or WBWB for females or ZAZA for males, for a theoretical maximum of 50% each. Exceptionally, instead of the WßWß or WBWB genotypes, some females became ZAWß following altered chromosome disjunction during meiosis [20]. To detect the presence of parthenogenetic or gynogenetic larvae, we first searched for the presence of WßWß or WBWB females. Then, as normal ZAZA males are indiscernible from the parthenogenetic or gynogenetic ones, we analyzed the possible deviations in the sex genotype ratio in the population using the peptidase-1 test. Before the space missions, the test was applied to all the females selected for the flight, to each of the males used for mating, and, after the flight, to the larvae born in space, when aged 2 or 3 mo (Table 2).
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Ground control progeny Twelve larvae developed from 1G-eggs obtained in the ground laboratory were derived from a ZAWB female previously inseminated by a ZAZA male. Four larvae were ZAWB genetic females and eight were ZAZA genetic males. No WBWB female was detected as expected for standard conditions.
Aboard µG-larvae A ZAWß female, preinseminated by a ZAZA male, generated 13 larvae that developed in µG up to stages 32–33. As the peptidase test showed, five larvae were ZAWß genetic females and eight were ZAZA genetic males. No larva had the WßWß genotype. Thirteen larvae, developed in µG up to stage 33, were derived from a ZAWB female crossed with a ZAZA male. Ten of them were ZAWB and three were ZAZA. No larva was of the WBWB female genotype.
Aboard 1G-larvae Three larvae, developed from eggs stored in the 1G centrifuge aboard station MIR, were derived from the mating of a ZAWß female with a ZAZA male. One larva was a ZAWß female and the two others were ZAZA males. No larva had the WßWß genotype.
In summary, 16 ZW females and 13 ZZ males developed from eggs fertilized under weightless conditions. The numbers of males and females were not statistically different from the theoretical or natural 50/50 male:female ratio [21]. These data and the cytological features of µG-embryos point out evidence that the aboard offspring resulted from an actual fertilization and not from gynogenesis.
Characteristics of Fertilization and First Cleavage Processes in µG
Cortical reaction of fertilization Eggs stored in µG for a minimum of 2 h underwent a cortical reaction, i.e., the migration of the cortical pigment toward the animal pole after fertilization, that demonstrated the success of egg activation in µG. However, both the cortical reaction and the egg activation process seemed to be altered. Aboard eggs exhibited large unpigmented areas at the sites of sperm entry, and the ascent of the pigment toward the animal pole was significantly amplified in comparison to control 1G-eggs (Fig. 2). In fact, it was still visible in µG-eggs up to the four-cell stage. Besides, no striking modification of the vitelline gradient was observed in cytological observations of the eggs of the 1G- and µG-replicates.
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Egg nucleus location Under natural conditions, the zygotic nucleus resulting from the fusion of the two pronuclei is located on the animal-vegetal axis, thus determining the meridian position of the furrow. Consequently, a furrow extended besides the animal-vegetal axis corresponds to the lateral position of the nucleus. Except for one, all examined eggs (n = 25) exhibited a normal location of the first furrow. This indicated that µG did not impair the migration of the pronuclei and did not provoke an off-axis location of the zygotic nucleus. This observation does not preclude a displacement of the zygotic nucleus from the animal–vegetal axis toward the vegetal pole, as observed in experiments using Xenopus in µG [6].
Evidence of effects of µG, gravity, and hypergravity on MV elongation In the present experiments, we compared the main characteristics of MV of eggs (n = 150) that were submitted to forces of µG to 3G during their first division cycle.
The MV, which were easily observed by SEM, covered the egg surface and displayed characteristic changes after fertilization. The MV transformations observed in the vicinity of the animal pole of P. waltl eggs were classified into five types corresponding to five stages of egg transformation during the first cycle [22]. At fertilization, referred to as stage 1, MV are hemispherical or conical-shaped and uniformly distributed (designated structural type 1). About 1 h after fertilization, MV progressively transform, organizing a discontinuous network of convoluted crests in association with deep depressions between 3–4 h postfertilization (type 2). That spongelike structure regresses from 4 h postfertilization (type 3). The egg surface is then smooth, exhibiting some flattened or spherical MV (type 4). At the beginning of the first division (stage 5, 6 h postfertilization) fingerlike MV elongate on the edges of the forming furrow (type 5, see Fig. 3), while on the other areas of the egg surface, MV are still of type 4. On the furrow base, between the furrow edges, rounded MV resembling type 4 MV develop.
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At fertilization, the conical MV had a uniform distribution and a density of 20 MV per µm2. Later, the number of MV per µm2 decreased but did not differ significantly between eggs submitted to different gravities. However, at the time of first cleavage the density of MV of 1.4G-eggs and 3G-eggs was strongly reduced, by four- and eightfold, respectively, by comparison to µG-eggs (Table 3).
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After 4 h postfertilization, a gravitational effect on MV became detectable. The values of MV diameters for µG- and 1.4G-eggs differed by more than 20% on average. Effects of gravity on 6-h-old eggs were more evident. The MV diameters progressively decreased by 25% from µG to 1.4G (0.40 µm and 0.32 µm for µG- and 1.4G-eggs, respectively). In contrast, it reached 0.87 µm for MV developed on 3G-eggs. That drastic increase in size resulted from the presence of very abnormal tufts of voluminous MV on the surface of the 3G-eggs.
Changes of the MV in shape and diameter seemed to be correlated to the gravity level applied to the eggs. At about 2–4 h postfertilization, the respective percentages of MV of the various types (round, ovoid, or elongated) were already different when eggs in µG up to 3G were compared. At stage 5, the difference in the patterns of MV of the furrow sides became more evident (Table 3). The percentage of rounded MV decreased by 30% from µG to 1.4G, whereas the percentage of elongated MV increased. It was 12% for µG-eggs and reached 38% for 1.4G-eggs. Finally, 96% of the MV of the 3G-eggs attained a typical voluminous lobed shape.
The MV appeared to be more sparsely distributed on the egg surfaces in 1.4G, 2G, and 3G than in µG or 1G. Comparing the two extreme cases of µG- and 3G-eggs proved very interesting. The percentage of MV that expressed a spatial distribution corresponding to an influence zone area of less than 2 µm2 was 100% in µG-eggs versus only 20% in 3G-eggs. The increase in MV size correlated with the increase in gravitational forces and roughly corresponded to an extension of the MV influence zone areas (Fig. 3 and Table 3).
Microvilli elongation was the most evident MV feature that appeared to depend on gravitational forces and on the time that these forces were applied to the eggs. Although the chronological schedule of MV pattern transformations occurred identically to that described for 1G-ground control eggs, hypo- and hypergravity constraints provoked alterations in MV characteristics (Table 3). As a rule, MV elongation, in reference to 1G-ground control eggs, was reduced in µG-eggs, was similar in 1G-eggs onboard the space station as well as in 1.4G eggs, and was amplified in eggs submitted to 2G and 3G. At about 4 h of development, µG-eggs displayed crests, 0.25 to 1 µm long, associated with cavities, 0.4 µm (±0.02) wide. In contrast, in 1G, ridges were about 1 µm and holes 0.6 µm (±0.09). They reached 1.5 µm or more in size, associated with holes of 0.65 µm (±0.06) and 0.75 µm (±0.07) in diameter, in 2G and 3G, respectively. Effects of gravity levels were also particularly clearcut for MV that extended along the furrow edges at about 6 h postfertilization. Comparing µG-eggs to 3G-eggs, these MV increased about fourfold in length (mean values: 0.6 µm and 2.0 µm, respectively, a statistically significant difference at the 0.01 level using Students t-test). The increase in size of the MV corresponded to the progressive emergence of MV of a length of 0.50 µm and more. Moreover, the size of the crests, i.e., periodic lobed structures resulting from fused MV, also reflected the effect of a long-term gravitational environment on MV pattern. The height of these crests (0.8 ± 0.2 µm in µG) increased threefold in 1G and more than fourfold in 3G-eggs. In these eggs, crests were considerably developed, forming prominent structures of 3 ± 0.2 µm elevation. Some of the MV developed up to more than 5 µm. The short fingerlike MV on the basement of the nascent first furrow also expressed a significant twofold increase in size (0.01 level of significance, Students t-test) when 1G- and 3G-eggs were compared (mean values: 0.12 µm and 0.22 µm for 1G- and 3G-eggs, Table 3).
DISCUSSION
Fertilization under µG Conditions
In P. waltl, activation of oocytes by electrical or thermal shocks or pressure on oocytes at the time of fertilization can lead to parthenogenetic animals [20, 23]. Moreover, gynogenetic larvae could also be obtained using the sperm of another amphibian species for egg activation. These animals only develop if a twofold increase in the chromosomes (diploidization) occurs as can be obtained under some experimental conditions [20, 24]. As nonfertilizing spermatozoa can induce activation of the oocytes, one of our goals was to provide evidence that animals obtained in space were the result of an actual fertilization process and not of a parthenogenetic or gynogenetic process. In P. waltl larvae, diploidy can be tested for using peptidase-1 as marker of the sex chromosomes and scoring the sex ratio. As shown in Table 2, no WW female, which would indicate gynogenesis, was detected, and no significant deviation of the percentages of females and males was observed in the animals obtained from fertilizations in space. So, these parameters indicated that no abnormalities of gene transmission, and consequently no induction of parthenogenesis or gynogenesis had occurred, strongly indicating that animals born in weightless conditions are the result of actual fertilization.
In addition, evidence of true fertilization was strengthened by the presence of spermatozoa in the perivitelline space and in the fertilization layer, and the presence of female and male pronuclei in the cytoplasm of the 2- to 6-h-old eggs. Additional features are the long-term development of animals born aboard the MIR station. Parthenogenesis or gynogenesis normally leads to lethality, whereas the percentages of developing individuals resulting from the groups of aboard animals and of ground control animals were similar. All previously published experiments on amphibian development in µG were conducted using the protocol of artificial fertilization, which is known as a possible source of chromosomal and genetic abnormalities. In our space experiments, fertilization occurred naturally: P. waltl oocytes were fertilized in the female genital tract by the spermatozoa stored in pelvic glands of the female. However, it should be noted that this normal fertilization occurred with gametes differentiated under 1G ground conditions. It is necessary next to demonstrate that male and female gametogenesis could occur in µG without any morphologic or genetic abnormalities.
Cellular Events of Fertilization in µG—Possible Roles for the Cytoskeleton
Microvilli patterning Microvilli elongation was clearly a main feature that seemed to depend on the gravitational environment. As a rule, MV alterations increased with the duration of exposure to a constant gravity force. A minimum of 2 h duration was necessary to induce an effect on MV in fertilized eggs. Thus, alterations were mostly observable at the time of first cleavage, which occurred 6 h after fertilization in ground conditions. Furthermore, some of the changes in the MV pattern appeared to depend on the magnitude of the gravitational forces, because MV elongation was gradually enhanced from µG to 3G. Actin is the basic cytoskeletal component forming the core of MV in eggs [25, 26]. Agents that favor actin polymerization or microfilament bundle formation also induce MV elongation [22, 27, 28]. In contrast, cycloheximide and cytochalasin B, which act by inhibiting actin polymerization and microfilament organization, provoke MV shortening [29–31]. Thus, MV changes appear to reflect the dynamic state of actin. Variations in the MV pattern from µG to 3G gravity environments suggest that gravitational forces could influence the spatial organization of the actin filaments in the amphibian egg and finally the dynamic state of actin. The fact that in µG, the organization of actin filaments in osteoblasts appeared modified and that the actin mRNA content in cells was depleted brings some support to such a hypothesis [32]. As various factors [27, 33, 34] are implied in the process of actin organization within a cell, further investigations are needed to specify which biochemical step is actually gravity dependent.
Egg activation and development During maturation of the amphibian oocyte and at the time of fertilization, the microtubular network of tubulin undergoes an important reorganization related to germinal vesicle breakdown, first polar body expulsion, and the cortical reaction of activation [35–38]. On P. waltl eggs, the identical aspect and location of the maturation spot and of the female nucleus, in µG- and 1G-oocytes prior to fertilization, suggest that changes in the microtubular system during maturation could occur in µG without much alteration. However, alterations in the pigmentation pattern of µG-eggs could reflect a differential sensitivity to gravitational forces of the microtubular system of eggs before and after fertilization.
The possible influence on embryonic development of these subtle subcellular alterations due to µG is becoming well documented. Xenopus eggs, fertilized in µG, established a normal bilaterally symmetric axis [39]. However, like embryos submitted to simulated weightlessness, embryos in µG displayed abnormalities in blastula and gastrula morphogenesis [40, 41]. In addition, in sea urchin embryos raised in µG, anomalies of the spicule pattern also seemed to result from an alteration of the primary mesoderm [42]. Though not demonstrated, these developmental changes could be attributed to an effect of gravity on the cytoskeleton of the cells implicated in early morphogenesis. These developmental changes could have resulted from an alteration of the microtubular system as suggested by in vitro experiments on microtubular dissipative structures [43]. In contrast, simulated hypergravity has also produced developmental disorders in the Xenopus egg attributed to alteration of the egg cytoskeleton [44, 45]. Animal cell lines when cultured in µG also displayed changes probably implicating the cytoskeleton [46–49]. Data obtained from cells of plants developed in µG indicated a major role for the cytoskeleton in response to gravitational force [50–53]. Summarizing these observations, it can be hypothesized that the cytoskeleton is the major target structure of the gravity effect at the cellular level. Nevertheless, in P. waltl, in spite of some cellular alterations induced in µG at the time of fertilization, embryos develop up to the adult stage. It seems that the well-known regulative process that characterizes amphibian development can compensate for possible intracellular disorders, ensuring apparently normal development.
ACKNOWLEDGMENTS
We are grateful to Christiane Tankosic, EA 2401, Henri Poincaré University-Nancy, and Monique Simon, Image Treatment Service and Inter-Universitary Center of Electron Microscopy, Paris, for their technical assistance. We thank Anne-Marie Duprat, Center of Developmental Biology, UMR 5547 CNRS, Paul Sabatier University Toulouse, for her contribution in the preparation of the scientific FERTILE experiment. We thank also the CNES board for its engineering and management, particularly Didier Chaput and Michel Viso, and the cosmonauts Claudie André-Deshays and Léopold Eyharts for their efficient practical expertise.
FOOTNOTES
First decision: 7 January 2000.
1 This work was supported by grants from the Centre National d'Etudes Spatiales, the Centre National de la Recherche Scientifique, and the Ministère de l'Education Nationale, de la Recherche et de la Technologie. 
2 Correspondence: Christian Dournon, EA 2401 Génétique et Interactions Cellulaires en Reproduction, Université Henri Poincaré, B.P. 239, 54506 Vandoeuvre-lès-Nancy cedex, France. FAX: 33 3 83 91 24 46; christian.dournon{at}scbiol.uhp-nancy.fr
Accepted: March 29, 2000.
Received: December 17, 1999.
REFERENCES
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  J. S. Tash, S. Kim, M. Schuber, D. Seibt, and W. H. Kinsey Fertilization of Sea Urchin Eggs and Sperm Motility Are Negatively Impacted under Low Hypergravitational Forces Significant to Space Flight Biol Reprod, October 1, 2001; 65(4): 1224 - 1231. [Abstract] [Full Text] [PDF]
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