European Space Agency

Fertilisation and Development of Xenopus Eggs on Sounding Rockets and in a Clinostat

G. A. Ubbels

Netherlands Institute for Developmental Biology, Hubrecht Laboratory, Uppsalalaan 8, Utrecht, The Netherlands

Introduction

Living organisms have developed special structures by evolution to perceive and cope with the gravity vector. In various organisms, the absence of gravity perturbs particular cell functions essential for normal embryonic development, such as cell proliferation and adhesion. Since classical embryological experiments1, 16, 17, 23 suggested that gravity is involved in determining the amphibian embryo's spatial structure, gravity may act as a signal in certain developmental processes, as do, for example, heat shocks at particular times in development. Microgravity shocks or longer periods of different gravity loads may influence normal development through, for example, perturbation of embryonic axis formation.

In the radially-symmetrical mature egg of Xenopus laevis the polar 'animal-vegetal' axis roughly foreshadows the embryo's main body axis. However, the egg needs some cue(s) for establishing additional polarities, prerequisite for normal development. As in many species, the sperm acts as such an additional cue in the, normally, monospermically-fertilised Xenopus egg.2, 23

Pigment concentrating around the Sperm Entry Point (SEP) marks the meridian that foreshadows the embryo's prospective ventral side. Since, in the majority of eggs, the blastopore forms at the meridian about 180° away from the SEP, the embryo's general body pattern is established from that time on. However, the dorso-anterior and ventro-posterior polarities can still be altered, by the influence of gravity and centrifugal forces, which cause the rearrangement of yolk components.2, 3, 7, 8, 23 This suggested that gravity in conjunction with the sperm establishes the dorso-ventral polarity. It was logical to test this hypothesis by experiments under µg during Spacelab flights 14, 16, 24, 25 and Sounding Rocket (SR) missions.4, 15, 16 for rev., 18-22, 26

Materials and methods

Biological materials
Eggs of the South African clawed toad, the anuran amphibian Xenopus laevis, are favourite subjects for developmental biologists. They are relatively large (~1.2 mm), easy to obtain and, because of their size, fairly easy to manipulate. As a result, much detailed information about the development is available (see refs. 6, 12, 16 for reviews). Over the years at the Hubrecht Laboratory, suitable female toads have been pre-selected and skin- marked.27 They could be relied on to provide sufficient numbers of eggs at predictable times and the eggs could survive the disturbances of launch and reentry, even after 24 hr of storage under Shuttle experiment preparation and launch conditions.14, 17, 24, 25

About four weeks before arrival of the toads at ESRANGE in Sweden, we set up a small amphibian facility with a simple water pumping system, thus filtering the circulating tap water and maintaining the temperature at around 22°C. The stress- sensitive toads were best transported packed in cooled insulated picnic boxes, partly filled with wet foam cuttings,24, 25 tranquillising them during the flight. Special care has to be taken to avoid mechanical shocks and large temperature variations. For the TEXUS 17 and MASER 3 missions, a private plane was hired to ensure the toads' safe arrival. Transportation to Kiruna by a commercial flight (as was done for MASER 6) is risky, particularly as the toads have to be transferred twice to another plane. Immediately on arrival, about three weeks before launch, about 30 Xenopus laevis females and about 40 males were placed separately into the aquaria. About a week later, the animals were rather well acclimatised and produced healthy eggs after hormonal stimulation under standard conditions of the Hubrecht Laboratory.

The eggs' restricted viability, the relatively long 'late access' period and the shortage of Payload Specialist time, especially at the beginning of Space Shuttle missions, prompted us to design an Automated Experiment Container (AEC) for the experiment on Spacelab IML-1.14, 26 This proved to be a real advantage when SR flight opportunities became available regularly. Hardware and live materials should be able to handle short periods of vibration, acceleration and deceleration that are intensified compared to those during Shuttle launches, but the much shorter late access period is an important advantage for performing biological experiments in µg. Unfortunately, as with Shuttle launches, there is the increasing tendency for lengthening the late access period for SR launches (Fig. 1).

Improved ESA technology
Fig. 1. Summary of experiments with Xenopus eggs in simulated and real microgravity. Line 1. Upon sperm penetration, the egg rotates as an entity in its capsule, turning its an-veg axis upright the yolky, heavy half down and the pigmented animal hemisphere up. Line 2. Experiments on clinostats were made after artificial fertilisation on the bench, or (Line 3) by fertilisation in the clinostat. Line 4. The experiments in real µg (~6 min), as on TEXUS 17, are handed over (HO) relatively shortly (1-2 hr) before Launch, though weather conditions or experiment malfunction can cause a delay (MASER 3, Line 5) or launch abort (as happened 6 days running with TEXUS 17). Line 6. MASER 6's eggs were fertilised under µg in flight; the first development of eggs was Line 7. Video-recorded shortly after fertilisation on Earth at L-5 min 30 s, from L-4 min to L-1 min, during L+73 s to L+419 s (the µg period) and after landing, 15-21 min after launch. Line 8. Fertilisation in a flight 1 g-centrifuge, simultaneously with fertilisation in µg, served as the proper control to discriminate whether any developmental anomaly found was due to a real µg effect. In addition, control eggs were fertilised after landing (see Fig. 5). Lines 9/10. Experiments in real µg on a Space Shuttle mission have to be delivered much earlier than for sounding rockets. For IML-1 and IML-2 it approached 15 hr before launch, and the gap is expected to become longer. Launch delays and aborts may also occur. Fertilisation in the Shuttle could be performed only about 7 hr after reaching µg, i.e. 24 hr after stripping eggs from the females (hardware loading inclusive).

Xenopus males and females were hormonally stimulated, precisely timed before a mission.15, 18, 19 The males were killed for removal of the testes and eggs were stripped from the females. Immediately after stripping, eggs from two of the usually four injected females were individually selected. Within 2 hr, the AECs were each loaded with salt solutions, histological fixative, one testis and two groups of 15 selected eggs from two different females. After stripping the same females for a second time, a second set of AECs was prepared for Earth control, to be activated 2 hr later. Selected eggs were also fertilised in some laboratory modules and dishes on the bench.

Hardware
The AEC (Fig. 2.1, Table 1) comprises a perspex cylinder block (79.5x19.0x33.1 mm, height with electronics 40 mm) equipped with spring-activated pistons for sequential operation, governed by a microprocessor.14, 16, 18, 26 In Shuttle flight experiments, each AEC was contained individually in a Biorack Type-IE container (Figs. 2.1 and 4) equipped with a connector for electrical activation and recording the temperature and housekeeping signals. 


Table 1. Activation programmes for the Automated Experiment
Containers on MASER 6 (time in seconds). 
------------------------------------------------------------
Onset 10 s after yo-yo despin

       T1   A1   R1   T2   A2   R2
P1     0    30   210   0   30   210
P2         (*)         0   30   210

       T    A     R    M   F1   F2
P3     0   30    60   170  210  -

------------------------------------------------------------
Onset 5-7 min before launch

       T1   A1   R1   T2   A2   R2
V      0    30   210   0   30   210
------------------------------------------------------------
F2 contained additional eggs and testis to perform manual
fertilisation after landing. *fertilisation on Earth (also see
Fig. 5)

Improved ESA technology

Fig. 2. Fertilisation of Xenopus Eggs: some Automated Experiment Containers (AECs). Fig. 2.1. AEC used on TEXUS 17, MASER 3 and Space Shuttle IML-1. 20, 24, 25 It consists of a perspex cylinder block (79.5x19.0x33.1 mm; 40 mm with electronics) equipped with spring- activated pistons governed by a microprocessor for sequential operation, providing fluid transfers between the different compartments of the closed system.18, 20, 26 Eggs were stripped from the females and selected. Within 2 hr, the AECs were loaded with salt solutions, histological fixative, a testis and 2x15 selected eggs. After another 2 hr and stripping the females for a second time, a second set of AECs was prepared for activation on Earth as a control; control eggs were also fertilised in some laboratory modules and in dishes on the bench. E: egg cylinder, and T: testis cylinder, both with 100% MMR and 2x15 eggs or one testis. A and R: cylinders with 10% MMR for fertilisation; M with 25% MMR for embryo culture; G with extra- contained glutaraldehyde fixative. Fig. 2.2.1. Modified AEC (mAEC) for the IML-2 Shuttle mission, providing improved circulation of fluids in the block, excellent visibility of two groups of embryos from different females for video-recording, in the now laterally positioned egg compartment (with two sub- divisions), and the possibility for an extra wash or post- fixation.4 Fig. 2.2.2. mAEC back view. Plungers, programmed for sequential activation to perform fertilisation are down (T, A, R), the additional three provide washing and the two histological fixations (M, F1, F2; not yet activated).4 Fig. 2.3. mAEC used on MASER 6, with two lateral egg compartments and two sets of plungers, independently programmable for fertilisation. Twice as many eggs could be fertilised in one go. However, histological fixation could not be performed in this module.4 The illustration shows how the left egg compartment is adapted for videorecording. The various inserts are interchangeable. 15 eggs from each of two different females and a testis were stored separately in the AECs, in 100% MMR (230 m Osmol and pH 7.8). The sounding rocket storage and fertilisation temperature was 18°C. Shuttle experiment storage was at 10°C; fertilisation in 10% MMR and culture at 22°C in 25% MMR. Eggs from only one female were available on MASER 6.

2.2.1 The 'Frog Eggs' experiment on TEXUS 17

In the 'Frog Eggs' experiment on TEXUS 17, eight AECs, each with its own temperature sensor, were housed together but individually electrically-activated in an air-tight double-walled aluminium box (Fig. 3.1- 3.3)15, 16, 18, 19, 26 specially designed and built by CCM (Nuenen, The Netherlands) as a subcontractor to ERNO (Bremen, Germany). The box provided excellent insulation for the flight duration and the conditions; the experiment units' internal temperature rose by only 1°C both in flight and on Earth; the pressure remained constant at 1 bar.15, 16, 26It was loaded aboard only 45 min before launch, inserted into TEM 06-15 (Fig. 3.4), a modification of TEM 06-11.21The autonomous power supply and the electronics package for recording housekeeping data, temperature, etc were mounted under the experiment platform. The AEC electronics and all the plungers (modified after the Spacelab D1 Shuttle mission) functioned flawlessly.

Improved ESA technology
Fig. 3. TEXUS module TEM 06-15 used for the fertilisation of Xenopus laevis eggs. Fig. 3.1. Air-tight double-walled aluminium box (120x150x350 mm). Fig. 3.2-3.4. 2x2 AECs were positioned along a horizontal bar plugged in on both sides of a midway vertical panel. The panel carried electrical connections on both sides for power supply and data recording (housekeeping signals and egg cylinders temperatures) from the eight experiment modules, which were individually activated. The autonomous power supply and the data-recording electronics package were positioned under the circular experiment platform (Fig. 3.4). Fig. 3.5. TEM 06-15 flew for a second time with Xenopus eggs on MASER 3. This time, the electronic unit was positioned in the box's front part.

2.2.2 The 'Frog Eggs' experiment on MASER 3
As on TEXUS 17, eight AECs were successfully flown in TEM 06-15, but the electronics serving these modules were centralised in the front part of the box 26 (Figs. 3.5 and 4), making the AECs cheaper and reusable. The mission was another successful flight test for the AEC.

Improved ESA technology
Fig. 4. Automated Experiment Container (AEC). See ref. 25. Fig. 4.1. AEC partially inserted into the Biorack Type-IE container, with incubator-A connector (c). Fig. 4.2. AEC without Type-IE container, as used on TEXUS 17 (see Fig. 2.1). Ct: wires connecting the microprocessor with the panel in the centre of the experiment box. Fig. 4.3. Mannually-operated flight-type laboratory module, without electronics. The plungers can be operated by turning the screws (s) on top.

2.2.3 The 'Frog Eggs' experiment on MASER 6
This time, the automated hardware was flown in Fokker Space's CIS-4 module (see Figs. 3.9, 3.12.c. and 3.13.a in Section 3 of this volume) and activated in two separate (0 g and 1 g) boxes. As on MASER 3, 26 the electronics serving the AECs was centralised. We used a modified version of the AEC (further denoted as mAEC 4 ; Figs. 2.2-2.3), which now contained two sets of three cylindrical holes, each set with an additional lateral egg compartment. The two sets were operated independently.

Thus fertilisation in each set could be performed at separately programmable times and twice as many embryos could be fertilised in one unit (Figs. 2 and 5). Furthermore, the modified arrangement of the different compartments improved the circulation of the various fluids in the mAECs and the visibility of the developing embryos in the units was excellent for video- recording (Fig. 2.3). However, histological fixation could not be performed in these modules.

In the mAEC's IML-2 version (Figs. 2.2.1. and 2.2.2)4 there was only one egg compartment and two of the six cylindrical compartments were available for histological fixatives. During the MASER 6 flight, two such modules were activated for an in-flight functional hardware test, including histological fixations. Both mAEC configurations performed well (Fig. 5).

Improved ESA technology
Fig. 5. Schematic overview of all MASER 6 experiment containers (with the mean of fertilisation rates per group of 15 eggs) fertilised in flight, either in µg (92%) or in the flying 1 g centrifuge (88%), and on Earth (72%). This time, eggs were used from one female because only one of the injected females produced good quality eggs, in a number just large enough to load 2x3 double and two single (IML-2) mAECs for flight and 1x3 double and one single mAEC for the ground control. Two mAECs were first loaded for video-recording in µg and on Earth, with eggs from the first strip. The ground control mAECs were activated 2 hr later than those in the two flight boxes. Because of poor handling during the air transfer, many of the pre- selected frogs died on the way to Kiruna and not many were left by the time of the fourth launch attempt. All mAECs therefore had to be loaded with eggs from only one female, and hardly enough good quality eggs were left for the ground controls, which is reflected in the total fertilisation scores. Moreover, one of the plungers failed in a ground mAEC, which is known to reduce the fertilisation rate. Only one of the cylindrical holes in the single mAECs (see Figs. 2.2.1 and 2.2.2) contained fixative. In the sixth cylinder there were full-strength MMR and 15 eggs and a testis, each in a separate membrane. Back in the laboratory, immediately after return from flight, the different groups of these eggs were fertilised manually with sperm flown in the same module. As an additional control, automated fertilisation was performed immediately after landing. In µg flight and in the ground control box (see Fig. 6), the cortical contraction was video-recorded in some of the eggs in one half of a double mAEC, which was provided with a special insert (Fig. 2.3) to keep the eggs in focus. As a control, the eggs in the other half of these mAECs were fertilised simultaneously with the recorded ones. Fertilisation rate variations could also be caused by differences in sperm fertility in the different testes.

Improved ESA technology
Fig. 6. Video-recording of the development of Xenopus eggs in µg. After fertilisation at L-5 min 30 s, recordings were made at L-4 min to L-1 min, in µg from L+75 s to L+419 s and after landing from L+15 min to L+21 min. In flight, n=3; on Earth, n=5. The figure shows only a very small temperature change, which cannot explain the very limited, but significantly earlier, start of the cortical contraction in flight (585 10 s after in-flight fertilisation, versus 679 9 s on Earth). The very small number of eggs recorded and the possible influence of launch disturbances prevent a firm conclusion. The minor, though significant, acceleration under µg of the cortical contraction initiation time prompts the suggestion of a reflight on MAXUS.

A 1g control centrifuge and a video recorder were used for the first time on MASER 6. Fertilisation was performed simultaneously under µg and 1 g conditions, so that flight perturbations other than varying g-loads were identical for all egg samples. Apart from the 1 g flight centrifuge, 1 g control embryos were obtained by activating a similar set of mAECs on Earth (Fig. 5). In both cases, the cortical contraction of fertilised eggs was recorded in the specially-designed facility; the eggs in the mAEC were held in focus with a special insert 4 (Figs. 2.4). Both of these newly-designed facilities, the flight centrifuge and the video recorder, functioned nominally.

After fertilisation of the different experimental groups in the mAECs activated on MASER 6 (Fig. 5) or after fertilisation in a fast-rotating clinostat (60 rpm), the embryos were carefully grown on Earth after retrieval and subsequently histologically fixed at different times. After staining the nuclei in the fixed blastulae, in optical serial sections of whole mounts analyzed by Confocal Laser Scanning Microscopy (CLSM),5, 16 we determined the number of blastomeres at the 8th cleavage and the shape and size of the blastocoel, using CLSM software for planimetry. In situ hybridisation analysis of whole mounts was applied for checking proper gastrulation in µg and in control embryos.4 Only eggs from one female were available for loading MASER 6's mAECs.

Biological results and discussion

'Frog Eggs' on TEXUS 17
Launched: 2 May 1988 (see references 15, 18-21, 16 for review) Crew: G. A. Ubbels, Principal Investigator (PI); J. Narraway (Hubrecht Lab). R. de Groot (Hubrecht Lab), H. van Soest (CCM); P. Kyr and P. Kuker (MBB/ERNO) for mission support.

Objectives: test the experiment container's plunger function under real flight conditions; determine whether Xenopus eggs could be fertilised during the brief µg period available (6-7 min); if so, whether under µg sperm penetrated only the animal hemisphere and whether the polyspermy- block, which at 1 g is caused by a transiently-enhanced egg membrane potential, also functions in µg.

Results and discussion
The eggs of a vertebrate, the anuran amphibian Xenopus laevis, were successfully monospermically fertilised and histologically fixed in µg for the first time. Analysis by normal histology and Scanning Electron Microscopy (SEM) revealed that, as in normal development, only one sperm penetrated somewhere in the egg's pigmented animal half. The polyspermy-block thus functioned as well in µg as under 1 g (Fig. 7). This was the first developmental biological experiment ever performed on a sounding rocket (SR). It demonstrated the feasibility of SR biological experiments in spite of the relatively high levels of disturbances (e.g. vibration, acceleration and deceleration) during launch and re-entry. SRs were thus shown to be appropriate vehicles for performing short-duration biological tests.

Improved ESA technology
Fig. 7. The single sperm penetrating the pigmented animal cap. 6 µm histological paraffin section. Fixation 138 s after fertilisation in the AEC during TEXUS 17. See ref. 18.

'Frog Eggs' on MASER 3
Launched: 10 April 1989 (see references 18, 20, 22, 26; 16 for review)
Crew: G. A. Ubbels, PI; J. Narraway and S. Kerkvliet (Hubrecht Laboratory); H. van Soest (CCM) and Dr. P. Kyr and P. Kuker (MBB/ERNO) for mission support.
Objectives: confirm the TEXUS 17 results, achieving fertilisation and histological fixation in µg at slightly different times; perform two pilot experiments: fertilise and fix eggs in an AEC that were first soaked in Hoechst 33258 (see below), and fertilise eggs in an AEC for live retrieval and careful culture on Earth.

Results and discussion
The MASER 3 experiment was a successful repeat of the TEXUS 17 experiment: sperm and eggs fused in-flight with a high score, even within the first minute.22 The eggs were successfully fertilised and histologically fixed in flight in one of the eight AECs after storage for almost 6 hr in Hoechst 33258. This is a rapidly penetrating DNA-specific bisbenzimide fluorochrome, which raised specific UV-fluorescence even after histological fixation. Both UV-fluorescence and subsequent SEM analysis 18, 20, 22 confirmed that sperm-egg fusion in µg starts real sperm penetration which, as shown later on IML-1, does indeed initiate embryonic development. In a second AEC, eggs again from two different females were fertilised but not histologically fixed. The embryos survived re-entry. They all developed normally but only until the gastrula stage before, depending on the egg sample, dying in the period between gastrulation and neurulation. Only four out of ten embryos from one female continued to develop, and these developed similar abnormal axes 18, 20, 22 (Fig. 8). It was thus concluded that a µg shock during fertilisation and/or initiation of development might perturb axis formation. The proper control, i.e. an onboard 1 g centrifuge, was not available at that time. However, a simulation experiment in laboratory modules mounted on a bicycle wheel 9 showed that the morphogenetic abnormalities found after fertilisation in the rocket were not due to variations in the g- levels simulated in that experiment and applied in a pattern identical to that on MASER 3.18, 20, 22 Vibrations were not included in this simulation. Eggs fertilised in MASER 6's 1 g flight centrifuge (see below) developed seemingly normally.4, 16

Improved ESA technology

Improved ESA technology

Improved ESA technology

Improved ESA technology
Fig. 8. Xenopus laevis embryos 5 days after fertilisation on MASER 3 (7 min µg, retrieval within 15 min). The embryos survived re-entry and gastrulated seemingly normally but, compared with the controls, development was retarded. They were slightly microcephalic and tail formation was strongly reduced. Analysis of 6 µm histowax sections (fixation in Bouin d'Hollande; alcoholic dehydration; azofuchsin, anilin blue, orange G stain; see ref. 23), showed that (bottom section) two notochords (1 and 2) had developed independently, the second apparently being induced by a secondary inducing centre. In a second larve (top section), the notochord (not shown) was split and tapetum tissue (a characteristic for eye development) was found in the bottom of the diencephalon (2; 1=normal eye). These developmental abnormalities could be explained if fertilisation in µg causes abnormal cell interactions later on, during gastrulation.22

Improved ESA technology

Improved ESA technology
Fig. 9 Normally-developing embryos were retrieved from MASER 6, after fertilisation in µg and in the flying 1 g centrifuge. We thus assume that formation of developmental anomalies in the MASER 3 pilot experiment (see Fig. 8 and refs. 18 and 22) were due either to using a different launcher or the inherent properties of egg samples produced by different females, and/or the 2.5 hr launch delay (see Fig. 1). Fig. 9.1. Five day- old abnormal Xenopus embryo from MASER 3 (see Fig. 8.) Fig. 9.2.1. Transverse 6 µm sections of a stage 42 12 Xenopus larve, grown on Earth after fertilisation on MASER 6 under µg, revealed only one normal, split notochord (n) and many other signs of normal axis formation. Fig. 9.2.2. Serial sagittal/longitudinal 6 µm sections through a larve from another egg, fertilised in the flying 1 g centrifuge on MASER 6, also showing normal morphology; for example, only one normal notochord (n).

'Frog Eggs' on MASER 6
Launched: 4 November 1993 (see references 4, 5, 16 for review)
Crew: G. A. Ubbels, PI; M. Reijnen and J. Narraway, (Hubrecht Laboratory); A. De Mazière (SRON/NWO), J. Gonzalez-Jurado (ESA). J. P. Leysten and A. Koppen (CCM) and Fokker Space for mission support.
Objectives: flight testing of design modifications of the IML-2 experiment unit (mAEC); flight testing of the newly- designed 1 g centrifuge and video-recording system; fertilisation of Xenopus eggs in the mAECs simultaneously at 0 g and in the 1 g centrifuge in flight; after retrieval and careful culturing of the embryos on Earth, they were histologically fixed at different developmental times. This could tell us whether a µg shock indeed perturbs axis formation and/or causes other developmental abnormalities (see the pilot experiment on MASER 3). Eggs were also fertilised and grown in a fast-rotating clinostat (60 rpm). We determined the number of blastomeres and the shape and size of the blastocoel, by using CLSM software for planimetry, in optical serial sections from whole embryos, fixed at the 8th cleavage, after staining the nuclei in whole mounts.8 In situ hybridisation on whole mounts was applied for checking proper gastrulation after fertilisation in µg and 1 g, and subsequent culture and fixation on Earth.4

Results and discussion
In the flight mAECs activated during MASER 6, eggs from two pre-selected females were fertilised at 0 g and in the 1 g centrifuge with equally high (80-98%) fertilisation rates (Fig. 5). After retrieval, they were grown on Earth and histologically fixed at different stages. The analysis confirmed normal axis formation (Fig. 8d).

In the MASER 6 embryos fixed at the 8th cleavage stage and after staining of the nuclei in whole mounts,5 we determined the blastocoel volume, the thickness of the blastocoel roof (in µm) in a 150 µm-diameter region around the animal pole, and the number of cells in the blastocoel roof that were hit by the animal/vegetal axis. These values did not significantly differ in the flight-embryos between 0 g and 1 g (Table 2). However, in the same embryos the ratio of the blastocoel floor level to the total embryo height was significantly different (Table 2 and Fig. 2 in ref.4). In embryos from eggs fertilised at 0 g the floor was consistently closer to the vegetal pole than in the embryos from eggs fertilised in the flying 1 g centrifuge. This effect is independent of the µg duration, as similar blastocoels were found in the embryos grown and fixed during IML-2 after the 24 hr-delayed fertilisation (Table 3). Nuclear counts in whole mounts of Xenopus blastulae, grown after fertilisation and fixed at the 8th cleavage on IML-2, also revealed no significant differences in mitotic rates (Table 3). Together with previous data from different cell systems, this suggests that, in a variety of cell types, mechanisms regulating mitosis are different or differently sensitive to µg. 


Table 2. Analysis of blastulae grown after fertilisation at
the beginning of 6 min of µg on MASER 6.

--------------------------------------------------------------------------------------------------------------------------

Mean±SEM                                            µg shock    1 g centrifuge     P

Blastocoel volume (in nl)                           41±8         45.0±:2.5         NS
                                                    (n=3)             (n=4)
Blastocoel roof(thickness in µm)                    258±12       247±13            NS
                                                    (n=4)             (n=4)
Number of cells in blastocoel roof hit by A/V axis  4.00±0.18    3.45±0.29         NS
                                                    (n=4)             (n=4)
Ratio of BC-floor level to embryo height            0.463±0.013  0.513±0.012     0.02<P<0.05
                                                    (n=4)             (n=4)
--------------------------------------------------------------------------------------------------------------------------
NS = not significant. SEM = standard deviation of the mean



Table 3. Analysis of blastulae grown in continuous µg on IML-2.


Mean±SEM                           µg       ground        P
-----------------------------------------------------------------------------
Total number of nuclei/embryo   333±28      346±18        NS
                                 (n=6)       (n=6)
 Blastocoel volume (in nl)       22±6        36±4         P<=0.05
                                 (n=7)       (n=9)
 Total embryo volume (in nl)    610±14      580±11        NS
                                 (n=7)       (n=9)
-----------------------------------------------------------------------------

NS = not significant

After fertilisation in the rotating tubes of a fast-rotating clinostat, i.e. in simulated µg, the embryos formed consistently significantly smaller blastocoels with a thicker roof than after fertilisation in the non-rotating tubes, i.e. at 1 g (Table 4). However, as after fertilisation in actual µg, there was no general increase in cell number in the whole blastula, nor an increase in the average cell number in the top segment only (Table 4). In blastulae from freshly stripped eggs fertilised in simulated µg, the blastocoel volume was slightly, but significantly, smaller than in the 1 g controls. At normal gravity, the blastocoel volume was not reduced by delayed fertilisation. However, the blastocoel was much smaller in simulated µg than in the 1 g controls, when eggs were fertilised after 24 hr storage at 10°C, as is routinely done in Shuttle experiments. Thus shorter as well as longer periods of actual and simulated µg affect proper blastocoel formation, probably through perturbation of fluid transport between the blastocoel and its surrounding cells.16 A lower position of the blastocoel floor after clinostatting is in agreement with results from previous experiments by others 10, 11, 28, 29 and, from experiments in actual µg,13, 16 which supports clinostatting as a suitable method, though not a replacement, for µg simulation experiments, even with the relatively large Xenopus eggs. 


Table 4. Blastulae developed in fast-rotating clinostat.

Mean±SEM                                µg           ground           P

---------------------------------------------------------------------------------------
Blastocoel volume (in nl)            24.8±2.4       33.3±2.2   0.01<P<0.02
                                      (n=11)         (n=12)
Blastocoel roof (thickness in µm)   204±6          152±6
                                      (n=11)         (n=6)
Cell number (whole embryo)          340±35         320±20           NS
                                      (n=6)          (n=11)
Cell number (top segment)            48±4           46±6            NS
                                      (n=11)         (n=6)
---------------------------------------------------------------------------------------
NS = not significant

Both the MASER 6 and clinostat experiments support the IML-2 results: the cleavage rhythm in Xenopus laevisis not influenced by µg. Moreover, in real and simulated µg, the blastocoel forms more centrally and vegetally, due to an enlargement of the roof cells and not to a general increase in cell numbers. In both clinostatted 10, 11, 16, 28, 29 and real-µg 4, 13, 16 embryos, these morphogenetic differences disappear in the late blastulae or early gastrulae. They do not interfere with mesoderm induction and normal development.4, 13, 16 Xbra in situ hybridisation on whole MASER 6 embryos, after fertilisation in flight at µg and 1 g, showed normal gastrulation.4 As the size of the entire embryo was unchanged, we assume that µg in some way interferes with the regulation of fluid accumulation in the blastocoel, due to changes in the osmoregulation governing the volume and composition of the blastocoelic fluid (see ref. 16 and its quoted references).

The cortical contraction,23 one of the first signs of successful fertilisation, was analysed by video-recording newly-fertilised eggs in flight and on Earth. After fertilisation 5 min 30 s before launch, the cortical contraction began in flight slightly earlier than on Earth (Fig. 6). Sperm penetration occurred on Earth in both groups, thus the differences in initiation times originated within the few minutes thereafter, and are probably not due to differences in the speed of sperm penetration on Earth. On Earth, at the same temperature, the entire process of contraction and relaxation takes about 12 min. This preliminary experiment should be repeated and extended, preferably on a longer flight such as MAXUS, so that a larger number of eggs can be recorded simultaneously and video-recording can be started in µg immediately after fertilisation (also in µg).

To date, the available data from developmental biological experiments on Earth and in space have shown that neither sperm nor gravity are required for embryonic axis formation in amphibians.16 Only Space Station experiments can tell us whether gravity determines polarity(ies) in the ovarian egg during maturation.

Acknowledgements

We gratefully acknowledge the skillful and enthousiastic support of our collegues at ESRANGE, ESA and ESTEC, as well as from MBB-ERNO during the TEXUS 17 and MASER 3 campaigns, and from CCM and Fokker Space. We gratefully acknowledge the financial support for the three sounding rocket experiments from ESA and the Dutch Organisation for Scientific Research (NWO, now SLW) through the Space Research Organisation of the Netherlands (SRON/MG-013).

References

  1. Ancel, P. & Vintemberger, P. (1948). Recherches sur le determinisme de la symetry bilaterale dans l'oeuf des Amphibiens. Bull. Biol. Fr. Belg. (suppl) 31, 1-182.

  2. Black, S. D. (1989). A step in embryonic axis specification in Xenopus laevis is simulated by cytoplasmic displacements elicited by gravity and centrifugal force. Adv. Space Res. 9, (11)159-(11)168.

  3. Black, S. D. & Gerhart, J. C. (1986). High frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage. Dev. Biol.116, 228-240.

  4. De Mazière, A., Gonzalez-Jurado, J., Reijnen, M. J., Narraway, J. & Ubbels, G. A. (1996). Transient effects of microgravity on early embryos of Xenopus laevis. Adv. Space Res. 17 (6/7)219-(6/7)223.

  5. De Mazière, A. M., Hage, W. J. & Ubbels, G. A. (1996). A method for staining of cell nuclei, in Xenopus laevis embryos, with cyanine dyes for whole mount confocal laser scanning microscopy.J. Histoch. Cytoch. 44, 399-402.

  6. Elinson, R. P. & Holowacz, T. (1995). Specifying the dorsoanterior axis in frogs: 70 years since Spemann and Mangold. Curr. Top. Dev. Biol. 30, 253-285.

  7. Gerhart, J., Ubbels, G. A., Black, S., Hara, K. & Kirschner, M. (1981). A reinvestigation of the role of the grey crescent in axis formation in Xenopus laevis. Nature 292, 511-516.

  8. Kirschner, M. W., Gerhart, J. C., Hara, K. & Ubbels, G. A. (1980). Initiation of the cell cycle and establishment of bilateral symmetry in Xenopus eggs. Symp. Dev. Biol. 38, 187-215.

  9. Morgan, T. (1904). The dispensibility of the constant action of gravity and of a centrifugal force in the development of the toad's egg. Anat. Anz. 25, 94-96.

  10. Neff, A. W., Wakahara, M. & Malacinski, G. M. (1990). Bifurcation of the embryo's axis: analysis of variation in response to egg centrifugation. Int. J. Dev. Biol. 34, 391-398.

  11. Neff, A. W., Yokota, H., Chung, H. M., Wakahara, M. & Malacinski, G. M. (1993). Early Amphibian (anuran) morphogenesis is sensitive to novel gravitational fields. Dev. Biol. 155, 270-274.

  12. Nieuwkoop, P. D. & Faber, J. (1975). Normal Table of Xenopus laevis (Daudin). 2nd ed. North-Holland Publ., Amsterdam.

  13. Souza, K. A., Black, S. D. & Wassersug, R. J. (1995). Amphibian development in the virtual absence of gravity. Proc. Natl. Acad. Sci. USA 92, 1975-1978.

  14. Ubbels, G. A. (1988). The role of gravity in the establishment of the dorso ventral axis in the amphibian embryo. In Biorack on Spacelab D1, ESA SP-1091. 147-155.

  15. Ubbels, G. A. (1988). A first fertilization of frog eggs in space. Microgravity News from ESA 2, 19-23.

  16. Ubbels, G. A. (1997). Establishment of polarities in the oocyte of Xenopus laevis. The provisional axial symmetry of the full-grown oocyte of Xenopus laevis. Cellular and Molecular Life Sciences 1, in Press.

  17. Ubbels, G. A. & Brom, T. G. (1984). Cytoskeleton and Gravity at work in the establishment of Dorso-ventral polarity in the egg ofXenopus laevis. Adv. Space Res. 4, (12)9- (12)18.

  18. Ubbels, G. A, Berendsen, W., Kerkvliet, S. & Narraway, J. (1989). The first seven minutes of a Xenopus egg fertilized on a Sounding Rocket in Space. In Microgravity as a tool in developmental biology. Special Symposium at the 11th International Congress of the International Society of Developmental Biologists, ESA SP-1123, 49-58.

  19. Ubbels, G. A., Berendsen, W. & Narraway, J. M. (1989). Fertilization of frog eggs on a Sounding Rocket in Space. Adv. Sp. Res. 9, (11)187-(11)197.

  20. Ubbels, G. A., Berendsen, W., Kerkvliet, S. & Narraway, J. (1990). Fertilization of Xenopus eggs in Space. In Proc. 4th Symp. on Life Sciences Res. in Space. ESA SP- 307, 249-254.

  21. Ubbels, G. A., Kerkvliet, S. & Narraway, J. M. (1990). Fertilization of Frog eggs in Space. In Texus 17-20 Abschluszbericht. 48-54.

  22. Ubbels, G. A., Berendsen, W., Kerkvliet, S. C. J. & Narraway, J. M. (1992). Fertilization and Development of Eggs of the South African Clawed Toad, Xenopus laevis, on Sounding Rockets in Space. Adv. Space Res. 12, (1)181-(1)195.

  23. Ubbels, G. A., Hara, K., Koster, C. H. & Kirschner, M. W. (1983). Evidence for a functional role of the cytoskeleton in determination of the dorsoventral axis in Xenopus laevis eggs. J. Embryol. Exp. Morph. 77, 15-37.

  24. Ubbels, G. A., Reijnen, M., Meijerink, J. & Narraway, J. (1994). Xenopus laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity. Adv. Space. Res. 14, (8)257-(8)269.

  25. Ubbels, G. A., Reijnen, M., Meijerink, J. & Narraway, J. (1994). The role of gravity in the establishment of the dorso- ventral axis in the amphibian embryo (05-NL-EGGS). In Biorack on IML-1. ESA SP-1162, 175-185.

  26. Ubbels, G. A. & Willemsen, H. P. (1989). Automatic experiment container (AEC) for fertilization of frog eggs in Space. A multipurpose piece of classified Space hardware. In Microgravity as a tool in developmental biology. Special Symposium at the 11th International Congress of the International Society of Developmental Biologists. ESA SP-1123, 75-78.

  27. Verhoeff-de Fremery, R. & Vervoordeldonk, F. J. M. (1982). Skin autografts as markers in the toad Xenopus laevis. Laboratory Animals 16, 156-158.

  28. Yokota, H., Neff, A. W. & Malacinski, G. M. (1992). Altering the position of the first horizontal cleavage furrow of the amphibian (Xenopus) egg reduces embryonic survival. Int. J. Dev. Biol. 36, 527-535.

  29. Yokota, H., Neff, A. W. & Malacinski, G. M. (1993). Analysis of experimental and quantitive parameters employed to assess the effects of novel gravity forces on the amphibian egg. Biol. Sci. Space 7, 85-100.

About| Search| Feedback
Right Left Up Home SP1206
Published April 1997.
Developed by ESA-ESRIN ID/D.