Sea Urchin Eggs under Microgravity Conditions

H.-J. Marthy

Observatoire Oceanologique, CNRS (URA 2156) - UPMC, F-66650 Banyuls sur mer, France

Early events in sea urchin development were successfully studied on three sounding rocket flights (MASER 4-6). The principle aim was the identification of potential effects of short exposure to microgravity on eggs at the fertilisation and cleavage stages.
An initial experiment on MASER 4 in 1990 suggested that fertilisation in µg would occur normally and that morphological aberrancies in advanced larval stages (plutei with strongly reduced arms) might be artifacts. The second experiment, on MASER 5 in 1992, conclusively confirmed and completed these results. There is no doubt that fertilisation sensu stricto does occur correctly in a monospermic way in the absence of gravity. Subsequent embryogenesis of such eggs on Earth leads to normal pluteus larvae. Surprisingly, virgin eggs could not be fertilised on the ground after exposure to µg.
The next experiment, on MASER 6 in 1993, studied whether cleaving eggs (early embryos) are affected by short exposure to µg. The results support the assumption that gravity changes are effectively sensed by the individual embryonic cells, but that development of the embryo as a whole is not affected. The working hypothesis is postulated for future experiments that the ability of embryonic cells in perceiving gravity is a cell cycle- related, or even a cell cycle-dependent, phenomenon (interphase or mitotic state). The experiments show that sounding rocket flights provide suitable opportunities for studies in developmental biology, with the proviso that precisely defined developmental events are selected as subjects of research.

Introduction

Studies on fertilisation processes have so far been performed aboard sounding rockets on two well known vertebrate and invertebrate egg models: the mesolecithal amphibian and the oligolecithal sea urchin egg. After the initial Spacelab D1 investigation¹ in 1985 into the fundamental question of whether µg affects the fertilisation process in the clawed toad Xenopus laevis, several sounding rocket experiments from 1988 onwards gave conclusive results.^2-8 The author reports that fertilisation sensu stricto and the establishment of the dorso-ventral polarity and of the bilateral body symmetry are not directly affected by µg. However, a causal relationship is likely to exist between µg exposure of the eggs during fertilisation and a morphological phenomenon at the gastrula stage (thickening of the epidermal blastocoelic roof) and some deformities (distorted reduced tail) appearing at the tadpole stage.

Thanks to flight opportunities offered by ESA, the substantial support of CNES and multi-purpose space hardware developed for the amphibian experiment,^{1, 9, 10} we were able to approach similar questions beginning in 1990 with another classic egg model: the sea urchin egg. In section 1, two experiments related to the fertilisation process are covered. Section 2 considers potential µg effects on embryos at cleavage stages.

1. FERTILISATION UNDER MICROGRAVITY CONDITIONS AND SUBSEQUENT DEVELOPMENT ON THE GROUND (MASER 4-5)

Major principles of fertilisation in animal organisms, including mammals, have been identified by studies on sea urchin eggs.^11-15 It was therefore a logical step to study its monospermic fertilisation under µg conditions. Any new result or observation from space experiments could be interpreted against a whole range of previous biological, structural, biochemical and molecular studies (for example, refs. 16-18). The eggs of the species Paracentrotus lividus (ripe in spring and in autumn) and Sphaerechinus granularis (ripe the whole year) appeared particularly well suited.

Materials and methods

Biological material
15 (MASER 4) and 30 (MASER 5) adult sea urchins (Paracentrotus lividus) were brought from the western Mediterranean Sea to ESRANGE. The animals were placed either individually or in small groups in plastic boxes filled with natural sea water. For transportation, several boxes were placed together in isotherm containers at about 16°C. The males and females were maintained separately in two aquaria at ESRANGE below 16°C to avoid accidental spawning.

Eggs and sperm were obtained by injecting the animals with either 0.2 ml of 0.1 M solution of acetylcholine chloride and sea water or by gently shaking. Sperm and eggs were released within about 2 min (Figs. 1-1 & 1-2) and collected separately in Petri dishes filled with filtered sea water. The Automatic Experiment Containers (AECs) for fertilisation studies ¹⁰ were loaded with pipettes from these dishes.

Fig. 1. The MASER 4 mission. Eggs and sperm are released (1-1 & 1-2) by adult sea urchins of the species Paracentrotus lividus after injection of a solution of acetylcholine/sea water. Eggs and sperm are separately stored and transferred to the various chambers of the experiment block shown in Fig. 1-3. The Automated Experiment Container (AEC), contained four identical fertilisation units. In activating the plunger of chamber 3, sea water was pushed, via the sperm chamber (2) towards the egg chamber (1), where the eggs were inseminated. In activating the plunger of chamber 4, a fixation solution was added to the egg chamber. Fig. 1-4. Launch of MASER 4 on 29 March 1992 at 10.07 local time. (Photograph reproduced with kind permission of P. Holm, Swedish Space Corporation.). Fig. 1-5. Basic post-flight observations on the eggs fertilised in µg. 5a: flown virgin eggs recovered live, no longer fertilisable. 5b: flown egg, fertilised and fixed during the µg phase; arrow indicates sperm attached to the fertilisation envelope. 5c: aberrant pluteus larva, differentiated on ground, from an egg fertilised in µg. The reduced arm growth is the most prominent defect; however, the larval body with a skeleton has been formed (arrow). 5d: young pluteus differentiated from a ground control egg, at day 7.

Experimental hardware and procedures
The AEC used for the MASER 4 experiment was a modification of the former multi-purpose space hardware developed by CCM (Nuenen, The Netherlands) for the amphibian experiment.⁹ This hardware (Fig. 1-3) was not totally satisfactory because of some toxicity in the latex membranes of the culture chambers. However, MASER 5's AEC (Fig. 2), specifically conceived and developed for sea urchin eggs,¹⁹ yielded excellent results.

Fig. 2. Automated Experiment Container (AEC). Fig. 2-1. Disassembled AEC hardware used on MASER 5. Photograph from P. Gerrits, Bergeyk, NL, reproduced with kind permission of CCM, Nuenen, NL. bl: experiment block. A plastic bag unit, filled with sea water (s), sperm (arrow) and a fixative solution (f), is inserted into the holes (s, e, f and arrow). Once the plastic covers (c) are placed on the six plastic bag units, the cover plate (co) and the mechanical-electronic plate (m) are attached. The plungers (p) are mounted on plate m. Unmarked pieces are used during the preparation procedures. The hardware is then integrated into the CIS boxes.¹⁰ Fig. 2-2. Assembled AEC flight hardware.

Loading the AECs was done 7 hr (MASER 4) and 10 hr (MASER 5) before launch. Insemination of the eggs on MASER 4 was performed automatically after 60 s of µg, followed by fixation of parts of the egg samples 60 s before the end of the µg phase, leaving 5 min of µg for the fertilisation process. On MASER 5, insemination occurred after 60, 300 & 360 s during the total of 420 s of µg, and the fixation plungers were activated for parts of the samples 30 s before the end of µg. For MASER 4-5, half of the egg samples fertilised during µg were recovered alive. In both cases, samples of virgin eggs and unused sperm were also recovered live.

Analysis
Initial post-flight examination of the eggs was possible 3.5 hr after lift-off (Figs. 1-4 & 3-1). Live recovered eggs were transferred to Petri dishes holding 5 ml of sea water and further development was recorded over 10 days (MASER 4) and 42 days (MASER 5). Fixed material was stored for structural analyses by Scanning and Transmission Electron Microscopy (SEM and TEM).

Results and discussion

Pre-flight tests
It was found²⁰ before the experiment that virgin eggs could best be stored at 5-11°C. The fertilisation rate was still high after about 12 hr, but it decreased rapidly after 24 hr. Dry' sperm stored at 5°C could be used for at least 48 hr. In a recent preservation study,²¹ we were able to prolong the storage time for virgin eggs and dry sperm at 4°C for more than 4 days.

Fertilised eggs exposed to 18 g hypergravity still showed essentially normal embryonic and larval development. Vibration tests indicated no harm to either virgin or fertilised eggs. As parthenogenesis was not triggered by vibration, it could be assumed that any effects on egg fertilisation and subsequent embryogenesis should be interpreted as a result of µg.

MASER 4 experiment (Fig. 1)
Based on elevation of the fertilisation envelope as the main gross morphological criterion for a successful fertilisation, only a low fertilisation rate of about 30% of the ground control samples and of 0-10% of the flight samples was found. The causes were a slight toxicity and delivery of the hardware only just before the campaign began.

These low percentages made a final conclusion difficult. However, SEM and TEM images (showing the presence of fertilisation envelopes, extruded cortical granules and elongated egg surface microvilli)²², showed that fertilisation under µg most likely happened in the normal monospermic way, triggering the whole cascade of fertilisation events (e.g. blocks to polyspermy, initiation of embryonic development). Several egg samples recovered live developed up to a pluteus stage, but showed a strongly reduced arm growth (Fig. 1-5c).²³

MASER 5 experiment (Figs.2 & 3)
The aim of this experiment was to confirm and complete the results from MASER 4 and, in addition, to identify possible µg effects on intact spermatozoa and virgin eggs. The hardware was completely redesigned for this purpose.¹⁹

The experiment was a complete success in both technical and scientific terms. The AEC worked nominally in space and on the ground at a constant temperature of 17°C inside the Cells in Space boxes.¹⁰ Analysis of live egg samples revealed a 95% fertilisation rate (Fig. 3.2-3.8), although the elevation of the fertilisation membrane was occasionally weak. The cleaving eggs continued embryonic and larval development. Young pluteus larvae were swimming after 4 days, identical to the controls. Larvae lifetimes could be increased to >40 days by feeding with microalgae. Interestingly, flown virgin eggs could no longer be fertilised. We have not pursued it being a µg effect. Flown sperm maintained its fertilisation ability on fresh virgin eggs. From the fixed samples, SEM and TEM studies confirmed successful insemination and fertilisation.²²

Fig. 3. The MASER 5 mission. Fig. 3-1. Launch of MASER 5 on 9 April 1992 at 12.07 local time. 317 km apogee, 7 min µg phase. Recovery of CIS-3 experiment boxes and payload occurred about 3.5 hr after launch. Fig. 3-2. Normal aspect of live eggs in translucent light: 2a. virgin eggs; 2b. left, inseminated egg surrounded by sperm (arrowhead); right, clearly fertilised eggs showing a fertilisation envelope (arrow). Diameter 80 µm. Fig. 3-3. Normal aspect of larvae at the pluteus stage, observed in translucent light. 3a: two swimming fully differentiated plutei at day 10. 3b: pluteus larva (a: anal arm with anal rod inside. b: body rod, or calcite spicule (arrow). e: oesophagus. i: intestine. o: oral lobe. s: stomach). Fig. 3-4. Immediate post-flight aspect of fertilised egg samples, recovered live. The eggs are at the cleavage stages. In total, about 40 000 eggs, fertilised in space, were recovered alive and continued development on the ground. An identical number served as ground control. All were from the same female and fertilised with the sperm of one male. Fig. 3-5. Eggs from another flown sample, at 8-16 cell stages of cleavage. No difference is visible between the various samples. Fig. 3-6. Egg fertilised (inseminated) after 60 s in µg and fixed after 5.5 min. A well-elevated fertilisation envelope surrounds the egg (arrow), formed by exocytosis of cortical granules within 60 s of the sperm-egg contact. It prevents polyspermy; the process is also known as slow block.' The presence of a fertilisation envelope is a clear indication of successful fertilisation. Fig. 3-7. Swimming larvae at the prism/early pluteus stage (about 40 hr old), differentiated on the ground from flown eggs. Fig. 3-8. Numerous normal young plutei at day 11, developed on the ground from flown eggs (compare with Fig. 3-3a/b). The developing larvae of flown eggs and of ground controls were cultured in small Petri dishes. From day 13 onwards, unicellular algae were offered as food. The last flown-egg pluteus, which showed no sign of metamorphosis, was lost at day 42.

Conclusion

Taking into careful account all observations on live and fixed eggs/larvae, some firm conclusions can be drawn:

• In sea urchin eggs, fertilisation under real µg conditions occurs in a normal monospermic way and with a high (normal) fertilisation rate.

• Eggs fertilised in µg develop normally on the ground to an advanced pluteus stage.

• The unspectacular results have to be interpreted in a positive way. It is reassuring to know that a classic animal egg model clearly shows that the initial crucial processes for creating new life are not disturbed in µg.

• Despite the brief duration of µg, sounding rocket flights are well suited to developmental biology studies, with the proviso that clearly defined processes are studied (e.g. fertilisation sensu stricto, a specific developmental step such as egg cleaving, and onset of gastrulation).

• It is an open question whether the well known and considerable regulation capacities of the sea urchin egg mask or compensate for some temporary µg effects at fertilisation during subsequent embryonic and larval development.

• It is also an open question whether eggs fertilised and kept under continued µg conditions would be able to develop normally. New space flight opportunities are necessary and therefore solicited.

2. GROUND-BASED DEVELOPMENT OF SEA URCHIN EGGS AFTER EXPOSURE TO MICROGRAVITY DURING CLEAVAGE STAGES (MASER 6)

Introduction
By selecting a precise portion of the development under study, the few minutes of µg available during a sounding rocket flight may serve for determining the potential effects of weightlessness and thus for determining the role of gravity on that particular developmental event.²⁴ Bearing in mind the step-by-step analysis of sea urchin development, the exposure of cleaving eggs to µg appeared not only worthwhile as the next step after the fertilisation study but also in the more general context of how cells might perceive gravity forces. In fact, an original hypothesis requires that the cell nucleus, anchored in the cytoplasm, mechanically interferes with the cytoskeleton and the cell membrane.^{25, 26} The slightest deviation in the cytoskeletal organisation by the nucleus, due to intracellular tension or pressure changes, would lead firstly to possibly irreversible modifications of the cytoskeleton and, ultimately, when the effect/s are amplified throughout succesive cell generations (as occurs in embryogenesis, for example), to severe morphogenetic disturbances. Yet a nucleus-cytoskeleton-membrane inter- relationship alone could hardly be the complete gravi-sensitive system.²⁷ The morphological situation had already been revealed to be more complex because the nucleus as a compact mass is present only during the interphase of the cell cycle but absent throughout mitosis. The question was therefore whether only interphase cells, and not mitotic cells, might be capable of perceiving gravity. In practice, the aim of the study was to check whether exposure of the cleaving eggs to a µg environment leads to a disturbed morphogenesis, and whether a disturbed morphogenesis results only when mg acts on interphase cells. The large translucent eggs in early, synchronously dividing, and in later, asynchronously dividing, cleavage stages appeared well suited to this purpose.

Materials and methods

For the MASER 6 flight (Fig. 4), cleaving eggs of the Sphaerechinus granularis sea urchin species were used. Artificially fertilised on the ground at 11°C, intact embryos at early and later cleavage stages were placed in the hardware developed by CCM (Figs. 4-3 to 4-5; ref. 19). Besides a passive static maintenance of parts of the samples, others were placed on a 1 g centrifuge. A sample of eggs in early cleavage was prepared for flight and ground video recording; the flight recording failed for technical reasons. Identical samples were prepared for ground controls. The total exposure to µg was 6 min. Parts of the samples were automatically fixed close to the end of the µg phase; others were recovered live and kept in culture for many weeks.

Fig. 4. The MASER 6 mission. Fig. 4-1. Launched on 4 November 1993 at 08.07 local time. 244 km apogee; 6 min µg phase. The CIS-4 experiment boxes were recovered after 1.5 hr. Fig. 4-2. Normal fully grown pluteus larvae of the sea urchin species Sphaerechinus granularis from eggs exposed to µg at the cleavage stages. Fig. 4-3. Two AECs with six culture chambers for a passive maintenance of about 1000 eggs/chamber. Fig. 4-4. Preparation of the 1 g onboard centrifuge (c) before insertion into the experiment CIS-box (e). Fig. 4-5. Assembled hardware for videorecording the cleaving eggs, before insertion into the CIS- box. Fig. 4-6. Post-flight MASER 6 with its integrated CIS-4 segment. Fig. 4-7. The first post-flight view of living eggs at their cleavage stages. These eggs developed into normal plutei larvae, as shown in Fig. 4-2.

Analysis

Post-flight analyses focused on the continued development in culture of the samples recovered live and on the quantitative estimation of the egg samples fixed at the end of the µg phase. In the first case it was expected that egg samples of early cleavage stages, after exposure to the few minutes of real µg, would develop into:

1. 100% normal larvae, meaning that there was either no effect or that all cells were only in mitosis, or

2. 100% abnormal larvae, meaning that there was either an effect on all cells or that all cells were simultaneously in interphase, or

3. into normal or abnormal larvae, meaning that there was an effect in one case (= interphase cells?) and none in the other (= mitotic cells?). From egg samples of late cleavage stages, it was expected that 100% should develop into abnormal larvae (simultaneous presence of interphase and mitotic cells).

Results and discussion

Whatever the cleavage stage exposed to µg, all eggs recovered live continued embryonic development and a high percentage (>75%) differentiated into normal plutei (Fig. 4- 2). The plutei were fed from day 12 with the microalga Isochrysis galbana (starvation occurs within about 3 weeks without feeding). The last larvae died after 2.5 months of culture, showing the first signs of metamorphosis.

How to interprete this clear result? Either there is no gravi- sensitive nucleus-cytoskeleton-membrane relationship conceivable on a mechanical basis, or there is such a relationship but the effects of brief gravity changes are regulated and development continues normally after return to normal gravity conditions. The quantitative exploration of the fixed material gave a clue that µg effects were produced (Fig. 5). In early cleavage samples from the onboard 1 g centrifuge (FCE), no advancement of the cleaving process was observed compared to the ground controls (GFE). There was a slight tendency in 0 g samples (FFE) towards more rapid cell division when compared to the GFE. It was clearer when compared to the FCE. Comparing later stages (asynchronous cell divisions), the 0 g samples (FFL) appear slightly advanced compared to those of the 1 g onboard centrifuge (FCL) and the ground control (GFL).

Fig. 5. Exposure to different g-levels (1 g ground, 0 g flight, 1 g centrifuge) of sea urchin embryos at early and late cleavage stages. Fixed early cleavage stages in 1 g (FCE), compared to ground control (GFE) and 0 g (FFE) samples, show no advancement of the cleavage process. A slight tendency for acceleration appears in 0 g samples compared to the ground controls. As to embryos at late cleavage stages, the samples in 0 g (FFL) appear to have a slight tendency for acceleration of the cleavage process, compared to the ground control (GFL) and the 1 g samples (FCL). Abbreviations NF: unfertilised eggs. F: fertilised eggs. 2C: 2-cell stage. 4C: 4-cell stage. 8C: 8-cell stage. MS: morulas. The evaluation considers only eggs recovered intact.

Conclusion

One main conclusion is evident from observations on the developing embryos and larvae: development of eggs exposed at cleavage stages to short µg conditions proceeds normally. Whether cleaving embryos in the interphase state or in mitosis were exposed, no long-term effects appeared during the subsequent embryonic and larval development on the ground. There were either never any effects or they became regulated throughout embryogenesis. Taking into account the quantitative results, this latter assumption has to be favoured. Whereas the embryo as a whole developed normally, the individual embryonic cells sensed apparent alterations in gravity and responded by shortening (1 g centrifuge) or lengthening (0 g) the cell cycle or a phase of it. Whether the interphase or the mitotic phase in embryonic cells will be revealed as the gravi-sensitive state is a challenging question for the future. The working hypothesis is postulated that the ability of embryonic cells (and possibly of other cell systems as well²⁸) for perceiving gravity is a cell cycle-related, or even a cell cycle-dependent, phenomenon.

Acknowledgement

Thanks are due to ESA for the flight opportunities and to CNES for substantial support. Particular mention must be made of Project Manager Mr W. Herfs at ESA/ESTEC and of his assistant Dr R. Demets (MASER 4 mission), as well of the mission scientists Mr J. Vreeburg (NLR Amsterdam, NL) and Prof G. Frohberg (TU Berlin, D). Various help is kindly acknowledged from Mr R. Huijser and Mr L. van den Bergh (Fokker Space, Amsterdam, NL), Mr H. Willemsen (CCM, Nuenen, NL) and his colleagues, as well as from Mr J. Zaar and S. Anflo, Project Managers of the Swedish Space Corporation. Last, but not least, many thanks to my team members P. Schatt and U. Marthy (Banyuls-sur-mer, F), who were directly involved in these projects.

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