SEARCH

SEARCH BY CITATION

Keywords:

  • development;
  • metamorphosis;
  • microgravity;
  • reproduction;
  • urodele amphibian

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Full development of a vertebrate during a continuous period has not yet been observed in microgravity conditions. In fish, mating and periods of development have been studied in Oryzias latipes during a National Aeronautics and Space Administration (NASA) shuttle flight (Ijiri 1997). In amphibians, embryonic development up to the hatching stage and periods of larval development have been observed in the anuran Xenopus laevis onboard a NASA shuttle (Souza et al. 1995) and on board a Russian Biocosmos satellite (Snetkova et al. 1995), respectively. The effects observed in X. laevis depended on the developmental stages at which the animals were launched and on the rearing devices used during the space flights. In birds, incubated quail eggs have been submitted to microgravity conditions in the Mir space station. Depending on the duration of egg incubation, the most quail embryos developed normally (Dadasheva et al. 1998; Barrett et al. 2000). In rodents, pregnant females have been maintained in microgravity during space flights of the NASA shuttles, but entire embryonic development has not yet been observed during a space flight (Kitajima et al. 1996; Serova et al. 1996).

After landing, the development of vertebrate embryos born and initially developed in microgravity has been studied in Oryzias latipes (Ijiri 1997) and in X. laevis (Souza et al. 1995). During the Cassiopée and Pégase French space missions, we have twice performed the so-called FERTILE experiment onboard the Mir space station. Our goal was to analyze microgravity effects on fertilization and embryonic development in the urodele amphibian Pleurodeles waltl. Actual fertilization in the absence of parthenogenesis or gynogenesis has been demonstrated. However, microgravity effects have been observed on the fertilized eggs during the first 6 h following spermatozoon penetration (Aimar et al. 2000). Further embryonic development occurred in microgravity conditions up to the hatching stage. Cell adhesion was altered during the cleavage and neurulation periods. Nevertheless, as a consequence of regulation phenomena, the live hatching larvae obtained at the end of the space flight had normal morphology and swimming behavior after the landing (L. Gualandris-Parisot et al., unpubl. data, 2001). In the present paper, we analyze development that occurred in the laboratory after landing in comparison with control ground-born animals. The progeny of animals that were born and initially developed in microgravity on board the Mir space station were analyzed up to the second generation. A particular interest was directed to the delayed metamorphosis only observed in animals from the Cassiopée space mission.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Animals

All P. waltl (urodele amphibian) used for the space missions derived from the standard strain of the Nancy laboratory. The animals were treated according to the French guidelines for the care and use of laboratory amphibians and according to the principles expressed in the Declaration of Helsinki. Embryos and larvae were staged according to the chronological table of development of the Pleurodeles waltl (Gallien & Durocher 1957).

In our laboratory, the period of reproduction occurs from September until March. From April to May, the males lose their sexual activity and regain it at the end of August, whereas the females can lay eggs all through the year.

Obtaining fertilized eggs in absence of males

To obtain fertilized eggs throughout the year, we used a double strategy based on a characteristic of urodele amphibians. During a natural mating, females take into the cloaca some spermatophores laid by males, ovulate and then lay fertilized eggs. However, the remaining spermatozoa are stored into the pelvic glands of their cloaca (Lemaitre-Lutz 1968; Sever 1992). Consequently, if egg laying is artificially induced a few months later, the remaining spermatozoa fertilize the oocytes in a natural manner in the absence of males. Thus, after natural breeding, such inseminated females guarantee that fertilized eggs can be obtained. However, the number of spermatozoa progressively decreases, particularly out of the reproductive period. To obtain fertilized eggs out of this period, we maintain the inseminated females at 8°C. With this method, we obtain fertilized eggs throughout the year.

Space experiments

The aim of the experiments was to study fertilization and embryonic development in P. waltl in microgravity conditions. Two repeated FERTILE experiments were conducted on board the Mir space station during the Cassiopée and Pégase French missions. Control experiments were synchronously performed in a ground laboratory.

Preparation of females Before each space mission, females were selected after a natural fertilization and classified using various criteria, such as weight, age, number of eggs laid, number of spermatozoa in the fertilization layer and perivitellin space, percentage of fertilized eggs and percentage of developed embryos. The first six selected females were put on board the Mir station and the next six were used as ground controls. The first experiment occurred during the 1996 Cassiopée mission from 17 August to 2 September and the second occurred during the 1998 Pégase mission from 29 January to 19 February. The Cassiopée mission occurred out of the natural reproductive period. Consequently, to keep the maximum of spermatozoa located in the cloacal glands alive after the natural mating, the inseminated females were maintained at 8°C for 12–16 weeks before the launch, in darkness except during maintenance. The Pégase mission occurred during the reproductive period. The inseminated females were maintained at room temperature (13–16°C) until the launch; that is, for 2–8 weeks after the natural mating. Before the space experiments, all inseminated females were fed with Chironoma plumosus and Tenebrio molitor larvae.

The dates of the first experiment were decided by the French and Russian space agencies. For the second space mission, we required our experiment to occur during the reproductive period.

The experiments For each FERTILE experiment, on board the Mir station as in the ground laboratory, six inseminated females received an intraperitoneal injection of 1.5 μL luteinizing hormone-releasing hormone (LH-RH; Sigma, St Louis, MO, USA; 10 μg/mL in physiologic medium) to induce egg laying, which occurred 25 ± 5 h later. On board Mir, as in the ground laboratory, eggs were collected and distributed in twin batches of ~20 eggs per batch. The first batches were stored on trays in ambient gravity: microgravity on board Mir and 1 g in the ground laboratory. The second batches were placed in a 1 g centrifuge that provided 1 g on board Mir and 1.4 g in the ground laboratory as a result of the centrifugal and gravitational forces in the control devices on the ground. Most embryos were fixed with a 2% formaldehyde solution at various stages of development and the rest were kept alive. The space missions were 16 and 21 days long, respectively. On board Mir, the embryos had the remaining 12 and 13 days, respectively, of development in microgravity. Both experiments were performed using the ‘Fertile’ instrument developed by the Centre National d’Etudes Spatiales (the French space agency; Gualandris-Parisot et al. 1998).

Larvae rearing conditions

Physiologic medium used to rear P. waltl embryos and larvae The physiologic medium used was initially described by Steinberg (1957), but was adapted for P. waltl. Two kinds of media were used, an operative medium and a rearing medium. The operative medium was composed of 340 mg/L NaCl, 100 mg/L KCl, 160 mg/L Ca(NO3).4H2O and 410 mg/L MgSO4.7H2O in sterile distilled water. The rearing medium corresponded to 10% diluted operative medium. For P. waltl, the pH of these solutions was adjusted to pH 7.4 using 56 mg Tris-HCl.

Larvae born in space In microgravity conditions, the embryos and larvae were reared at 18°C up to hatching in sterilized fresh water from Nancy. During the first week after the landing, the young larvae were individually reared in the modified Steinberg operative medium that was progressively diluted during the second week with modified Steinberg rearing medium. The larvae were reared for 1 week in this rearing medium and then placed in fresh water and reared under standard laboratory conditions. The control progeny derived from the control ground-born females were reared under the same conditions. The Pleurodeles larvae were fed with nauplii of the crustacean Artemia salina prepared in the laboratory.

Larvae reared under standard laboratory conditions To investigate the development of the larvae born during the Cassiopée mission, the variation in development duration from laying to metamorphosis was studied in 25 offspring obtained in different seasons. These progeny were reared in standard laboratory conditions; that is, in fresh water, in plastic basins, at room temperature and submitted to the daily variation of light. The temperature was registered for 3 years.

Peptidase-1 assays

Peptidase-1 is a marker that allows the identification of the different sexual genotypes of P. waltl. Two codominant genes encode this dimeric and polymorphic enzyme: (i) pep-1A, carried by sex chromosome Z; and pep-1B or pep-1β, carried by sex chromosome W. The males have a ZAZA genotype and the females a ZAWB or a ZAWβ_ genotype (Ferrier et al. 1980,1983; Dournon et al. 1988). Each genotype gives a specific electrophoretic pattern on a starch gel. The ZAZA males are characterized by one slow homodimeric band (aa) and the ZAWB (or ZAWβ) females by three bands: (i) a fast homodimeric band (bb or ββ); (ii) a medium heterodimeric band (ab or aβ); and (iii) a slow homodimeric band (aa). Previous experiments performed on board the Russian satellite BION 10 have indicated that the electrophoretic pattern of peptidase-1 is not modified by a 12-day orbital journey and that peptidase-1 remains a reliable sex marker (Bautz et al. 1996b).

The peptidase-1-test is based on the detection of peptidase activity. In order to ascertain the sexual genotype of larvae, tail tip fragments were homogenized as previously described (Rudolf & Dournon 1996). The homogenates were analyzed by electrophoresis on starch gels followed by peptidase-1 activity detection according to the method Lewis and Harris (1967) modified by Nicholson and Kim (1975). The peptidase-1 hydrolyzes specifically the dipeptide valyl-leucine that is used as a substrate in the assay reactions (Rudolf et al. 1997).

After the two space experiments, peptidase-1 assays were done twice on the live larvae born on board the Mir station and the ground control larvae. In order to ascertain whether the sexual genotypes diagnosed by the peptidase-1 test agreed with sexual phenotypes, the gonadal sex of each animal was identified by dissection or laparotomy at the adult stage.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Development of embryos and larvae on board Mir and in the ground laboratory during the space missions

On board the Mir station, 10 of the 12 inseminated females provided embryos, which developed in microgravity and in 1 g. Among the 12 ground control females, 11 provided embryos developed in 1 g and in 1.4 g ground devices. The percentage of development calculated for the two space missions was defined as the number of developed embryos at the 2-cell stage or later stages divided by the total number of eggs. The percentage of developed embryos was 21% and 82% for the eggs laid by the microgravity on-board females and 23% and 78% for the eggs laid by the 1 g ground control females, for the Cassiopée and Pégase missions, respectively. The percentage of developed embryos was 13% and 87% for the 1 g centrifuged eggs on board Mir and 10% and 73% for the 1.4 g centrifuged eggs in ground laboratory, for the Cassiopée and Pégase missions, respectively (Table 1). The development time from laying to hatching was the same for the animals reared at 18°C on board the space station as in ground laboratory. The results presented in the present paper do not include the activated eggs fixed before the 2-cell stage that were used for studying fertilization in microgravity (Aimar et al. 2000).

Table 1.  Percentage of developed embryos laid on board the Mir space station or in the ground laboratory and the number of animals kept alive after landing and crossed when mature
  % DevelopedNo. larvae keptNo. live larvae usedNo. reproductive
 Total no. eggsembryosalive after landingfor peptidase-1 assaysadults
First mission: Cassiopée
On board Mir
In microgravity (= 0 g)5042122139
In 1 g centrifuge (= 1 g)14713333
On ground
In ground device (= 1 g)52723000
In 1 g centrifuge (= 1.4 g)13710000
Second mission: Pégase
On board Mir
In microgravity (= 0 g)7198229135
In 1 g centrifuge (= 1 g)17387000
On ground
In ground device (= 1 g)68078661212
In 1 g centrifuge (= 1.4 g)12573000

Concerning embryonic development, preliminary results showed that in microgravity conditions the cell adhesion was altered. During the cleavage period, the intercellular space was enlarged in surface area and during the neurulation period cells came off the neural plate (Fig. 1a–d). The analysis of embryonic periods is in progress. Although the precocious embryonic development was not strictly normal, as a consequence of embryologic regulation phenomena, the tail-bud embryos had a normal morphology. Moreover, the live hatching larvae obtained after landing had normal swimming behavior. Using the Fertile instrument, a video movie taken on board the Mir space station clearly confirmed that embryos with such abnormalities developed into hatching larvae.

image

Figure 1. Embryos during the cleavage period (a) in microgravity conditions (bold arrow, abnormal cleavage; thin arrow, separation between the top of the blastomeres) and (b) in the ground laboratory. Bar, 0.5 mm. Embryos during the neurula period (c) in microgravity conditions (arrow, isolated cells) and (d) in the ground laboratory. Bar, 0.6 mm.

Download figure to PowerPoint

Number of larvae reared in the laboratory after the space missions

Cassiopée mission At landing time, 22 larvae that had developed in microgravity up to hatching stages 31–32 were kept alive and transported for rearing in the Nancy laboratory. Unfortunately and without correlation with the space flight, nine young larvae died. Thirteen larvae underwent peptidase assays to determine their peptidase-1 and sexual genotypes. Five larvae were genetic ZAWβ females and eight were genetic ZAZA males.

Three larvae developed in the 1 g centrifuge on board Mir up to stages 32–33 were kept alive and then reared in the laboratory. One larva was a genetic ZAWβ_ female and two others were genetic ZAZA males. All of the ground embryos were fixed as controls and consequently no control larva were obtained for this experiment (Table 1; Fig. 2).

image

Figure 2. Electrophoretic patterns of peptidase-1 indicating the ZAWB female and ZAZA male sexual genotypes of larvae born in microgravity. At the left, indicated in bold type is the pattern of the standard ground female.

Download figure to PowerPoint

Pégase mission At landing time, 29 larvae developed in microgravity were kept alive. They were at the hatching stages 32–33 as the ground control ones. They were separated into two batches and reared in the laboratories of Nancy and Toulouse. Unfortunately and without correlation with the space flight, 16 young larvae died. Thirteen larvae underwent peptidase assays. Ten larvae were genetic ZAWB females and three were genetic ZAZA males. No larvae were obtained alive from the 1 g centrifuge because all of the obtained embryos were fixed during the flight.

At the landing time, 66 ground control larvae developed in 1 g were kept alive. They were separated into two batches and reared in the two laboratories. Many young larvae died because of a rearing incident and only 12 larvae underwent peptidase assays. Four larvae were genetic ZAWB females and eight were genetic ZAZA males. No larva were obtained alive from the 1.4 g ground experiment (Table 1; Fig. 2).

Development from hatching stage up to achieved metamorphosis

Cassiopée mission After recovery in the laboratory, the microgravity and 1 g larvae born in space had similar development duration and body length. Compared with the data of the table of development at 18°C (Gallien & Durocher 1957), development of these larvae was similar up to larval stage 54 and increased up to stage 56, when metamorphosis was achieved. At this stage, the animals were 8 months old (6–9 months), instead of 3.6 months (3.3–4 months) (Fig. 3a), and were 110 mm (85–116 mm) in length instead of 72 mm (62–82 mm; Fig. 4). For this experiment, no ground control larva were obtained alive after the landing time. Because direct comparison was impossible, we reared numerous progeny in the following years and particularly at the beginning of the reproductive period.

image

Figure 3. Duration of the larval development up to metamorphosis for (a) progeny born onboard the Mir station during the Cassiopèe mission (—) and standards according to the table of development at 18°C (–-; Gallien & Durocher 1957). (b) Natural progeny (–-) laid before the Pègase mission and synchronous 1 g ground-control progeny (—) laid during the Pègase mission (same female). (c) Natural progeny (–-) laid before the Pègase space mission and born-in-space progeny (—) laid during the Pègase mission (same female). Bars indicate the maximum and minimum values of the samples. Depending on the rearing temperature, all progeny were comparable in development for the Pègase mission, but not for the Cassiopèe mission.

Download figure to PowerPoint

image

Figure 4. Larvae at the beginning of metamorphosis (stage 55). Larvae born and reared up to the hatching stage onboard the Mir space station during the (a) Cassiopèe and (b) Pègase missions, then reared in laboratory conditions. (c) Ground control larva at the same developmental stage. Bar, 20 mm.

Download figure to PowerPoint

Pégase mission After recovery of the larvae in the laboratory, the development of the microgravity animals born on board Mir and the 1 g ground control animals was similar until metamorphosis. No abnormal growth was observed (Fig. 4). This development was comparable with the development of progeny derived from the natural crosses obtained before the space mission with the same females (Fig. 3b,c). Moreover, this development was also comparable with that of progeny reared at the laboratory during the corresponding annual period (Fig. 5).

image

Figure 5. Mean duration of development up to achieved metamorphosis and mean length for 25 natural progeny derived from standard females during the reproductive period. Apart from temperature, which varied according to the seasons, the rearing conditions were the same for all the progeny.

Download figure to PowerPoint

Juvenile development and sexual maturity of animals born in space

After metamorphosis, the animals grew without observable abnormalities.

For the Cassiopée mission, 12 months after the landing, 10 males had callosities and turgescent cloacal lips. Five months later, the six other animals acquired the female phenotype. For the Pégase mission, 11 months after the landing, three males had callosities and turgescent cloacal lips. Four months later, the 10 other animals differentiated into females. The sexual phenotypes of the ground controls became evident during the same periods. For the both missions, the 29 mature animals, 13 males and 16 females, had a sexual phenotype in accordance with the sexual genotype diagnosed from peptidase-1 assays during the larval development.

In November 2000, nine males (seven microgravity and two reared at 1 g in flight) and three females (microgravity) from the Cassiopée mission were still alive and were 47 months old. One male (microgravity) and four females (microgravity) of the Pégase mission were still alive and were 31 months old.

Analysis of offspring derived from animals born in space

When a male is in the presence of a female for reproduction, the sexual parade normally occurs, but in some cases, the female does not lay eggs. In these cases, the operator does not know whether the male is functional or not and whether the female is inseminated or not. For these reasons, each male that was born and developed up to the hatching stage in microgravity was first crossed with a standard ground female. If the female laid fertilized eggs, the same male was crossed for a second time with a female born and developed in space.

Seven microgravity males were crossed with nine standard ground females (Table 2). Only four females laid fertilized eggs. In the second mating, three of these seven males were crossed with the three females that were born and developed up to the hatching stages in microgravity. These three females laid fertilized eggs. In the third mating, one of these three latter males was crossed with a female that was born in microgravity but developed in the 1 g centrifuge on board Mir. This female laid fertilized eggs. Other crosses were performed without success.

Table 2.  Results of crosses between ground control animals and/or animals born and developed to hatching on board Mir during the Cassiopée mission
Crosses between:    %% 
Females born in spaceMales born in spaceGroundGroundNo.F2 offspringFertiliz-Develop-% Abnormal
1 gμG1 gμGfemalesmaleseggsref.ationmentembryos
  1. *Abnormalities expressed during cleavage stages and previously observed in standard progeny, abnormalities expressed at tail-bud stages. Sd, standard animal; UF, unfertilized eggs; μG, microgravity.

  A96/301 β96/21 0
  A96/303 β96/27 0
   A96/304β96/22 0
   A96/304β96/35 453A-9892871
   A96/305β96/25 0
   A96/305β96/29 0
   A96/306β96/18 4B1a-981001000
   A96/306β96/18 647B1b-9892912
 β96/315 A96/306  604B2-9886852
β96/302  A96/306  871B3-98918927*
   A96/310β96/31 997C1-9893901
 β96/319 A96/310  399C2-988682< 1
   A96/309β96/24 201D1-988779< 1
 β96/316 A96/309  546D2-989393< 1
   A96/312β96/36 422 UF
   A96/318β96/14 0
    Sd femaleSd male279c-978987< 1
    Sd femaleSd male348g-979592< 1
    Sd femaleSd male199h-979391< 1
    Sd femaleSd male703j-979897< 1
    Sd femaleSd male1634a-989695< 1
    Sd femaleSd male425b-989189< 1
    Sd femaleSd male961f-9892911
    Sd femaleSd male1426g-9897961
    Sd femaleSd male165h-98888718
    Sd femaleSd male793j-989897< 1

For all progeny, the percentages of fertilization (number of embryos at the 2-cell stage/total number of eggs) and development (number of embryos at the tail-bud stage/total number of eggs) were in accordance with those obtained for standard progeny in our laboratory (Table 2). Moreover, the morphology of these larvae, which were observed at periodic intervals during embryonic and larval development, was without any particular distinguishable abnormality. The duration of development until metamorphosis was achieved was comparable with that of progeny obtained and developed during an identical annual period (Table 3; Fig. 5). No abnormality was detected in animals reared until adulthood. Some animals are now sexually mature and fertile. The others are becoming mature.

Table 3.  Larval development of progeny (F2) derived from one or two parents born on board Mir during the Cassiopée mission
  Time to metamorphosisTime toNo.
F2 offspringLaying datefor first metamorphosedmetamorphosis for 50%metamorphosedRearing
ref.(year/month/day)animal (weeks)of animals (weeks)animalsconditions
  1. Details of the parents of these progeny are given in Table 2. t°, Temperature.

A-9898/02/23253234Ambient t°
B2-9898/02/23253213Ambient t°
C1-9898/02/23263532Ambient t°
D1-9898/02/23252926Ambient t°
B3-9898/03/11223131Ambient t°
D2-9898/03/24213419Ambient t°
C2-9898/03/24253114Ambient t°
B1b-9898/05/0719225Ambient t°
B1b-9898/05/07152525Ambient t°
B1b-9898/05/0752526Constant 18°C,
     in darkness

Survival of the 12 females put on board the Mir station

Of the six females of the Cassiopée mission, one of the five females that laid eggs on board the Mir station died during the second month after the landing. This female had begun to get thin during the space flight. In November 2000, three females were still alive. Of the six females of the Pégase mission, three are still alive. The death of the females could not be correlated with space flight constraints.

Post-flight progeny of the adult females

Four embarked females were crossed with ground standard males. Post-flight progeny were obtained and the percentages of fertilization and developed embryos agreed with those obtained for standard progeny. No particular abnormalities were observed.

Influence of temperature and the seasons on larval development

Temperature in the laboratory rearing room For 3 years, the maximum and minimum water temperatures were measured in the rearing basins and the mean temperature calculated ((minimum + maximum)/2) for each week. The maximum amplitude of the mean temperature was observed each year between August and December averaging 11–12°C. Immediately after the warm period of summer, the temperature decreased rapidly (Fig. 6).

image

Figure 6. Annual statement of temperature in the rearing room of the laboratory. For 3 years, the range of the water temperature was measured each week. The maximum amplitude of temperature recorded each year was ~11–12°C and occurred between August and December.

Download figure to PowerPoint

Standard progeny reared under laboratory conditions Immediately after the observation of delayed metamorphosis in the Cassiopée animals, we studied the development of 25 natural progeny derived from standard females; that is, without cold treatment or hormonal laying induction. Under laboratory conditions, the duration of development from egg laying until metamorphosis depended on the seasons of laying and rearing. The progeny laid at the beginning of the reproductive period had the longest duration of development, at 43 weeks, whereas the progeny laid at the end of the reproductive period had the shortest, at 19 weeks (Fig. 5). The metamorphosed animals in these two groups of progeny (N = 81 and 72) were 69 mm in mean length (range 52–95 mm), and 59 mm (range 53–63 mm), respectively. In each group of progeny, the larvae did not homogeneously develop. Some animals that were too skinny could not metamorphose and were eliminated.

Moreover, in the same B1b-98 progeny reared in daylight and at ambient temperature (Table 3), the duration of development up to metamorphosis of five larvae individually reared in 100 mL water and a batch of 25 larvae reared in 2500 mL water was 22 and 25 weeks, respectively. For the same ratio of the number of larvae : volume of water, the duration of development until metamorphosis was comparable. However, in darkness and at a constant temperature of 18°C, the duration of development of six larvae from these same progeny individually reared in 100 mL was 52 weeks.

Progeny derived from cold-treated and hormonal laying-induced females The aim was to verify whether both the cold treatment applied to inseminated females before the mission and the injection of hormone could be involved in the delay of metamorphosis of the larvae born in space during the Cassiopée mission. The development of progeny of five inseminated females reared at 8°C over 23–35 weeks was analyzed. One week after return at room temperature, all females were stimulated by an LH-RH injection and laid fertilized eggs that developed into larvae. Three females laid eggs in September; that is, at the beginning of the period of reproduction (Table 4). Their progeny achieved metamorphosis after a 34–37-week development. The metamorphosed animals (N = 40) were 79 mm in mean length (range 71–86 mm). One female laid in November and the progeny metamorphosed after a 31-week development. The metamorphosed animals (N = 9) had a mean length of 60 mm (range 45–70 mm). The fifth female laid in May; that is, at the end of the reproductive period. The animals (N = 53) metamorphosed in 21 weeks. The length was not measured. No influence of either the cold treatment or the hormonal injection was detected.

Table 4.  Duration of development to metamorphosis for hormone-induced progeny derived from cold- or non-cold-treated females
 Conditioning of femalesProgeny development
 CoolingRearing time Time toTime to
 time beforeat RT before metamorphosis formetamorphosis forNo.Mean
Ref. forinjectioninjectionDate of layingfirst metamorphosed50% of animalsmetamorphosedlength in
females(in weeks)(weeks)(year/month/day)animal (weeks)(weeks)animalsmm (range)
  1. Progeny were reared under the annual temperature conditions in the rearing room. RT, room temperature.

Cold-treated females and hormone-induced progeny
D-9832198/05/07102153
E-97 P23297/09/1729341080 (78–82)
A-97 P28197/09/2330361278 (71–85)
C-97 P25297/09/2532371878 (71–86)
D-97 P35197/11/273031960 (45–70)
Non-cold-treated females and hormone-induced progeny
O-9898/02/0822231056 (50–70)
Q-9898/02/082222955 (50–60)
P-9999/09/0736412473 (57–95)

Progeny derived from hormonal laying-induced females not treated with cold Three progeny derived from three inseminated females reared at room temperature were obtained by LH-RH injection. One was obtained at the beginning of the reproductive period and the other two at the end of this period. These progeny achieved metamorphosis after 41 and 22–23 weeks of development, respectively. The metamorphosed animals (N = 24 and 19) had mean lengths of 73 mm (range 57–95 mm) and 55 mm (range 50–70 mm), respectively (Table 4). No influence of the hormonal injection was detected.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

For the first mission, our goal was to verify that factors other than microgravity could modify the rate of the larval development of the Cassiopée mission animals after their return on earth. For the second mission, we clearly showed that after fertilization and disrupted embryonic development in microgravity conditions, further development to adulthood and reproduction occurred after landing without any detectable abnormalities.

Influence of temperature and season on larval development

In heterothermal species such as P. waltl, the duration of development varies depending on ambient temperature. At low or high temperatures, the duration increases or decreases. In the laboratory rearing room, the temperature of the rearing water varied depending on the season, with a maximum amplitude of 11–12°C between August and December. For all studied offspring, the duration of development from the hatching stage to metamorphosis depended on the seasons in which larval development occurred. In laboratory conditions, progeny laid between January and March developed and achieved metamorphosis more quickly than progeny laid between September and November. The durations of embryonic and of larval development were ~19 weeks and ~43 weeks, respectively. Curiously, metamorphosis of progeny laid between January and March occurred before the metamorphosis of progeny laid between September and November. This result does not only show that the decreased rearing temperature induced an increase in development duration, but also that this decreased temperature, when applied during larval development, maintained the slowing down of development over a long period.

Both cold treatment of females before launch and hormonal injection for egg laying did not delay the occurrence of metamorphosis in progeny. The duration of development of such progeny was in accordance with that of standard offspring. As in standard offspring, the length of larvae was higher when the duration of development increased and shorter when the duration decreased. Moreover, depending on the rearing temperature, it appeared that the light could also play a role in the duration of development (Table 3).

The animals of the Cassiopée mission were born in August; that is, at the end of the reproductive period, and were reared when the ambient temperature was decreasing in the rearing room. According to the table of development, the duration of development and the size of these animals were increased. The animals of the Pégase mission were born in February; that is, during the reproductive period, and were reared when the ambient temperature was increased in the rearing room. The duration of development and the size of these animals were normal. For both the Cassiopée and Pégase animals, the duration of development up to metamorphosis was comparable to that of control animals reared during the same annual periods at ambient temperature. The sizes were also comparable. The variation in the duration of development is clearly due to the variation of the ambient temperature during the rearing period. However, an additional factor was the variation of the natural light period. The variation in the length of the larvae also had an explanation. The larvae increased in size with an increasing duration of development. This phenomenon has been well described in anurans (Decker & Kollros 1969; Dournon & Chibon 1974), but not in P. waltl. Nevertheless, we have previously observed delayed metamorphosis for animals embarked at the neural stage 15 for a 12-day space flight on board an automatic Russian satellite called Photon 10 (Bautz et al. 1996a). After landing, two live larvae that had developed in the satellite up to larval stage 32 were reared under laboratory conditions. During the space flight, the first animal was under microgravity conditions and the second one was at 1 g using a centrifuge on board the satellite. These two larvae died before the achievement of metamorphosis. They were 8 and 14 months old and 54 and 113 mm in length, respectively (Bautz et al. 1996a). They were derived from a female that had laid eggs in February after a hormonal injection. The delay of metamorphosis in these two animals has no explanation, even if their abnormal length is explained by the duration of the larval period.

Development of and reproduction in animals born in space

The percentages of development of embryos and larvae on board the Mir station and in the ground control laboratory during the both space missions were consistent with the criteria for the selection of females for the space flights. The females were selected after a natural mating depending on the number of spermatozoa in the perivitellin spaces and fertilization layers of their eggs (Aimar et al. 2000). The six females with the highest number of spermatozoa were selected for the space mission and the others remained on the ground as controls. Consequently, the eggs laid by the ground control females had a lower percentage of development, particularly the eggs laid during the first mission, which occurred out of the reproductive period. However, the good and comparable yield of developed embryos on board Mir and in the ground laboratory indicated that the conditions in microgravity were appropriate for development. The duration of development from laying until the hatching stages was the same on board the space station and in the ground laboratory at 18°C.

After landing, the behaviour and morphology of animals born in space during the Cassiopée and Pégase space missions seemed normal. During all of development up to adulthood, we detected no abnormalities that could distinguish these animals from standard animals. The analyses of their offspring showed that the percentages of fertilization and development were in accordance with the control animals. The duration of development until metamorphosis was in accordance with that of control offspring, as well as the duration of development of the larvae deriving from the Cassiopée progeny with the ground controls obtained later (Figs 2,5). No genetic abnormalities were detected during the analysis of offspring.

To verify a hypothetical effect of microgravity on sex reversal, the sexual genotype was determined using the peptidase-1 test and the gonad sexual phenotype was determined using dissection or laparotomy. No sex reversal occurred under the influence of space flight. This point was verified because P. waltl can reverse sex due to epigenetic effects, such as rearing temperature (Dournon & Houillon 1984).

After landing, the adult females that had laid fertilized eggs on board the Mir station were not perturbed by the space flight. The oldest females, those from the Cassiopée mission, are 7 years old at present. Most standard females from the rearing are younger. The good health of the Cassiopée and Pégase females is explained by the particular care lavished in the technical assistance of the laboratory.

In conclusion, the on-ground development of the animals born in space and the development of their progeny showed no differences with control P. waltl animals. Depending on the methods and limits of the analyses, the results clearly indicated that these amphibian animals born in space were able to live and reproduce after returning on earth. These results totally agree with those obtained with the fish Oryzias latipes (Ijiri 1997). Fish, and now amphibians, open the way for the colonization of space by man. Now, our present objective is to verify whether animals that are born in microgravity and reared up to sexual maturity on board a space station can reproduce without negative consequences after their return on earth.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the Center National d’Etudes Spatiales (CNES) board and particularly Didier Chaput and Michel Viso for engineering and management. We thank the French cosmonauts Dr Claudie André-Deshays and Lieutenant-Colonel Léopold Eyhartz for their efficient, practical expertise in the experiments FERTILE-1 and FERTILE–2, respectively. We are grateful to our Russian colleagues, particularly Rocket Kosmic Korporation Energia colleagues, for the preparation and success of the space missions. This work was supported by grants from the French space agency CNES.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • Aimar, C., Bautz, A., Durand, D. et al. 2000. Microgravity and hypergravity effects on fertilization processes in the salamander Pleurodeles waltl. Biol. Reprod. 63, 551558.
  • Barrett, J. E., Wells, D. C., Paulsen, A. Q., Conrad, G. W. 2000. Embryonic quail eye development in microgravity. J. Appl. Physiol. 88, 16141622.
  • Bautz, A., Durand, D., Oukda, M., Tankosic, C., Dournon, C. 1996a. Les missions spatiales 1995 et 1996 du laboratoire de biologie expérimentale-immunologie de l’université Henri Poincaré de nancy. Bull. Acad. Soc. Lorr. Sci. 35, 195201.
  • Bautz, A., Rudolf, E., Mitashov, V., Dournon, C. 1996b. Peptidase-1 expression in some organs of the salamander Pleurodeles waltl submitted to 12-day space flight. Adv. Space Res. 17, 271274.DOI: 10.1016/0273-1177(95)00645-u
  • Dadasheva, O. A., Gur'Eva, T. S., Sychev, V. N., Jehns, G. 1998. Characteristics of morphogenesis of the Japanese quail embryos during microgravity. Aviakosm. Ekolog. Med. 32, 3841.
  • Decker, R. S. & Kollros, J. J. 1969. The effect of cold on hind-limb growth and the lateral motor column development in Rana pipiens. J. Embryol. Exp. Morphol. 21, 219233.
  • Dournon, C. & Chibon, P. 1974. Influence de la température, de lâge et des conditions hormonales (thyroxine) sur la prolifération cellulaire chez la jeune larve et pendant la métamorphose du Crapaud Bufo bufo L. (Amphibien Anoure). Roux Arch. Entw. Mech. Org. 175, 2747.
  • Dournon, C., Collenot, A., Lauthier, M. 1988. Sex-linked peptidase-1 patterns in Pleurodeles waltlii Michah. (urodele amphibian): genetic evidence for a new codominant allele on the W sex chromosome and identification of ZZ, ZW and WW sexual genotype. Reprod. Nutr. Dev. 28, 979987.
  • Dournon, C. & Houillon, C. 1984. Genetic demonstration of functional sex inversion in Pleurodeles waltlii Michah (Urodele Amphibian) under the effect of temperature. Reprod. Nutr. Dev. 24, 361378.
  • Ferrier, V., Gasser, F., Jaylet, A., Cayrol, C. 1983. A genetic study of various enzyme polymorphisms in Pleurodeles waltlii (Urodele Amphibian). II. Peptidases: Demonstration of sex linkage. Biochem. Genet. 21, 535549.
  • Ferrier, V., Jaylet, A., Cayrol, C., Gasser, F., Buisan, J. J. 1980. Etude électrophorétique des peptidases érythrocytaires chez Pleurodeles waltlii (Amphibien Urodele): mise en évidence d’une liaison avec le sexe. C. R. Acad. Sci. Paris Sèrie D. 290, 571574.
  • Gallien, L. & Durocher, M. 1957. Table chronologique du développement chez Pleurodeles waltlii Michah. Bull. Biol. Fr. Belg. 91, 97114.
  • Gualandris-Parisot, L., Bautz, A., Chaput, D., Husson, D., Durand, D., Dournon, C. 1998. Mises au point technologiques en vue d’étudier le développement du Pleurodèle (Amphibien Urodèle) à bord de la station spatiale MIR. Récents Progrès en Génie des Procédés 62, 3748.
  • Ijiri, K. 1997. Mating behavior of the fish (medaka) and development of their eggs in space. Biol. Sci. Space 11, 153167.
  • Kitajima, I., Semba, I., Noikura, T. et al. 1996. Vertebral growth disturbance in rapidly growing rats during 14 days of space flight. J. Appl. Physiol. 81, 156163.
  • Lemaitre-Lutz, F. 1968. Anatomie des glandes pelviennes de la femelle de Pleurodeles waltl Michah., leur rôle de réceptacle séminal. Ann. Embryol. Morphol. 1, 409416.
  • Lewis, W. H. P. & Harris, H. 1967. Human red cell peptidase. Nature 215, 351353.
  • Nicholson, J. A. & Kim, Y. S. 1975. A one step L-amino oxidase assay for intestinal peptide hydrolase activity. Anal. Biochem. 63, 110117.
  • Rudolf, E. & Dournon, C. 1996. Activity of peptidase-1, a sex linked enzyme, during larval development at normal or sex reversal rearing temperature and according to tissues in adults of Pleurodeles waltl (urodele amphibia). Comp. Biochem. Physiol. 115, 177186.
  • Rudolf, E., Girardet, J. M., Bautz, A. M., Dournon, C. 1997. Purification and partial characterization of peptidase-1, a sex linked enzyme in Pleurodeles waltl (urodele amphibian). Biochem. Cell Biol. 6, 803806.
  • Serova, L., Natochkin, I., Nosovskii, A., Shakhmatova, E., Fast, T. 1996. Effect of weightlessness on the mother- fetus system (results of embryological experiment NIH-R1 aboard the ‘Space Shuttle’). Aviakosm. Ekolog. Med. 30, 48.
  • Sever, D. 1992. Comparative anatomy and phylogeny of the cloacae of salamanders (Amphibia: Caudata). IV. Salamandridae. Anat. Rec. 233, 229244.
  • Snetkova, E., Chelnaya, N., Serova, L. et al. 1995. Effects of space flight on Xenopus laevis larval development. J. Exp. Zool. 273, 2132.
  • Souza, K. A., Black, S. D., Wassersug, R. J. 1995. Amphibian development in the virtual absence of gravity. Proc. Natl Acad. Sci. USA 92, 19751978.
  • Steinberg, M. 1957. A non-nutrient medium for culturing amphibian embryonic tissues. In Carnegie Institution of Washington Year Book, Book 56, p. 357. Carnegie Institution, Washington (DC).