The mating process of Chlamydomonas eugametos was studied under microgravity conditions on a sounding rocket. Gamete suspensions of opposite mating type were combined and then chemically fixed after 0, 2,4 & 6 min of µg. A synchronous reference experiment was run on the ground at 1 g.It was determined that, in µg, sexual activation of the gametes was less than at 1 g. However, indications were found that the gametes' diminished performance in the rocket was not due to the µg conditions, but to the launch environment.
The mating process of the unicellular green alga Chlamydomonas eugametos is a well-researched example of cell-to-cell interactions. C. eugametos produces two opposite types of gametes, referred to as mt+ and mt- (mt stands for mating type). Whereas mt+ and mt- gametes are morphologically identical, they carry different adhesion molecules called agglutinins at the surface of their flagella (Fig. 1). The mt+ and mt-agglutinins are mutually adhesive and it is by means of these compounds that mt+ and mt- gametes are able to recognise each other, bind, and present one another with the signals required for a co-ordinated execution of the mating process, which culminates in sexual cell fusion. The early steps of the mating process include a steep increase in the adhesiveness of the flagellar surface, effected by aggregation of agglutinin molecules, and a transformation of the flagellar tips that comprises tubulin polymerisation. Under 1 g conditions, a substantial rise in adhesiveness and a significant activation of the flagellar tips can be measured within 6 min, which is the µg experiment window offered by the MASER sounding rocket.
Fig. 1 A suspension of gametes of C. eugametos. The gametes come in two opposite mating types that are morphologically identical. The cells are propelled by two frontal flagella. The oval-shaped cell body is about 8 µm long.
Cell cultures Mt+ and mt- gamete suspensions of C. eugametos were obtained by flooding 2-3 week-old agar cultures with 20 ml Hepes buffer at pH 6. Typical density of the suspensions was 107 cells/ml.¹
Experiment hardware: flexible tube units
The experiment units used were flexible tube units (see Section 3.3 of this volume). Four flexible tube units were exposed to µg, with a reference set of four identical units exposed to 1 g on the ground. Each unit contained eight tubes, each with three compartments for mt+ gametes, mt- gametes and a fixing solution (2.5% glutaraldehyde).² By adding Percoll, the buoyant density of the gamete suspensions was carefully equalised to that of the fixing solution to ensure that mixing of the fluids would take place at equal rates in µg and 1 g.¹ In order to create the correct conditions for sexual cell fusion, the gamete suspensions were illuminated by red and green LEDs.¹
Tumbling table: prevention of sedimentation at 1 g
It was found during preparatory ground tests that the mating process could be accelerated by preventing the cells from sinking to the bottom of the culture vessel.¹ This could be achieved simply by inverting the vessel every 20 s. This gravity effect would undoubtedly influence the outcome of the sounding rocket experiment, as sedimentation does not occur in µg. To eliminate this unwanted effect, the 1 g reference samples were inverted every 20 s on the 'tumbling table' tilting platform (Fig. 2), manually operated on the ground throughout the 6 min µg exposure of the flight samples.¹, ²
Fig. 2 The 'tumbling table', a manually-operated tilting platform that was crucial for the 1 g reference experiment on the ground (see text).
Bio-assay for flagellar adhesiveness
The adhesiveness of the flagella is preserved after chemical fixation. To quantify the adhesive power of the flagella, fixed and washed samples are presented with live gametes of opposite mt, which quickly bind to fixed partner cells. If the fixed material is diluted in, for example, 2-fold steps, each following dilution elicits a weaker binding response until, finally, there is no longer any binding. The last dilution step that still provoked binding was used as a semi-quantitative indicator. The adhesiveness was expressed as 2x , with the '2' denoting 2-fold dilution steps (also referred to as 'titer') and the exponent 'x' expressing the ultimate positive dilution.
Reduced flagellar adhesiveness
By comparing flown samples fixed at 0, 2,4 & 6 min after mixing mt+ and mt- cells, it was unequivocally established that the gametes had been sexually activated in µg. Between t=0 and t=6 min the flagellar adhesiveness had grown progressively stronger (Fig. 3) and, likewise, the flagellar tips had been significantly activated.² The conclusion is that Chlamydomonas gametes are capable of mating under µg conditions. Remarkably, the flagellar adhesiveness in the µg-exposed samples was consistently lower at all points in time, including t=0 (Fig. 3). This suggested that, in the flight series, the gametes had lost part of their flagellar adhesiveness before t=0. As the flight and ground series had been sampled from the same stock suspensions, and since the environmental conditions of the flight and ground series had been identical up until launch, the reduction of adhesiveness could have been caused by two possible events: the launch of the rocket, or the brief exposure to µg (duration: about 5 s) preceeding t=0.
Fig. 3 Evolution of flagellar adhesiveness in the rocket and on the ground. The adhesiveness was determined at four points in time after mixing mt+ and mt- cells. The experiment was performed in quadruplate. Each bar represents the mean value and standard deviation of the flagellar adhesiveness of four individually mixed and fixed samples. Two different mt+/mt-combinations (I and II) were tested, derived from batch cultures of different age.
The second hypothesis was tested in October 1989 during ESA's 8th parabolic flight campaign, 6 months after the sounding rocket experiment. In parabolic flight aboard the Caravelle, Chlamydomonas gametes were exposed to short periods of µg (0-15 s).³,4, 5 No indications at all were found that the adhesiveness of the flagellar surface was affected by brief exposures to µg. Having found no evidence in support of the second hypothesis, we were left with the first as a more plausible explanation.
The 75 s boost phase of the MASER sounding rocket is immediately followed by 6 min of µg. During launch, the test samples are exposed to vibrations, accelerations and acoustic noise at levels that can affect a living system.5 Therefore, if an organism behaves unusually after launch, this does not automatically mean that it has reacted to µg. Instead, it might be an after- effect of a disturbance introduced during launch. We suppose that the latter explanation applies to our experiment. The occurrence of this artifact could not have been be predicted. Using ground- based test equipment, the separate influences of launch vibrations, linear accelerations and noise were investigated by the authors but significant effects were not identified.¹, 5 Apparently, for biological materials such as Chlamydomonas gametes a realistic launch simulation required imposing these three elements in concert. However, a test facility for this did not exist.
1 g reference centrifuge
Onboard 1 g centrifuges were introduced in the sounding rocket programme after our experiment had been completed. Having onboard 1 g reference samples may have helped us to remedy the unequal flagellar adhesiveness at t=0. On the other hand, it is difficult to conceive how elimination of sedimentation (a special requirement in our experiment) could be fulfilled on a centrifuge.
Is the Chlamydomonas mating process sensitive to
Handicapped by the unequal adhesiveness at t=0, the results obtained do not allow us to decide whether the Chlamydomonas mating process is influenced by µg or not. Our conclusion is that the gametes were able to mate under µg conditions, but did so at a slower rate, apparently due to a launch effect.
The authors would like to thank the following persons and organisations for their support: Louis Aartman (NLR), Wolfgang Briegleb (DLR), Lennart Dreier (SSC), Dirk Frimout (ESA/ESTEC), Ulf Högman (Esrange), Ron Huijser (Fokker Space & Systems), Antoon Koppen (CCM), Dick Mesland (ESA/ESTEC), Alan & Loeki Musgrave (University of Amsterdam), Jan Rietema (CCM), Sven Wallin (SSC), Harry Willemsen (CCM), Jan Zaar (SSC), CEV, CNES, Orbitics and Ibis Hotels. The experiment was funded by SRON.
The five publications listed below are directly related to the described Chlamydomonas sounding rocket experiment. All are recommended for further reading, in particular the detailed 'Unicellular algae in space' trilogy. For information about the biology of the Chlamydomonas mating process, the reader is referred to the literature lists contained in these articles.