The aim of this study was to discover the motility differences of frozen and thawed bull spermatozoa under weightless and ground conditions. Significant differences were found using a computerised sperm motility analyser. The analysis showed alterations in straight line and curvilinear velocity, as well as in linearity values. The amount of progressively motile spermatozoa, including all spermatozoa with velocities >20 µm/s, increased significantly from 24±9.5% in the reference test, to 49±7.6% in the µg test. In conclusion, there is strong evidence that gravity influences sperm motility.
Several experiments and studies have shown that some processes occur differently and more favourably under µg than under terrestrial conditions. Consequently, certain properties can be produced only under µg.
In this study, the motility of frozen and thawed bull spermatozoa was investigated under weightlessness and compared with motility under normal conditions on Earth. The influence of gravity on functional processes of higher biological systems was thus studied. For many reasons, the spermatozoon is an ideal model to examine physiological functions, e.g. the study of motility. It remains unknown to what extent and manner biological functional processes such as cell differentiation, transportation and development are influenced by gravity. Investigations performed under µg should therefore contribute to the understanding of the genesis, structure and function of gravity- perceiving cells, and should elucidate the importance of gravity on molecular mechanisms involved in basic biological functions. The effect of gravity on sperm movement characteristics was clearly demonstrated in this investigation.
The great interest in recent years in the objective analysis of sperm motility has made it increasingly apparent that progressively motile spermatozoa and their movement characteristics are of biological and, hence, clinical importance.
Semen samples were obtained from bulls at the Grub animal breeding and insemination station in Germany, collected by an artificial vagina. Samples showing at least 80% sperm motility were diluted to a concentration of 40- 50x106/ml and frozen in 0.5 ml straws according to the method described in ref. 1. The frozen samples were thawed and maintained at 39°C until examination.
Experimental design and mission
The experiments were carried out in November 1988 (TEXUS 19) and May 1990 (TEXUS 26). The samples were measured in a specially developed airtight and constant- temperature (39°C) chamber (Strömberg-Mika, Bad Feilnbach, Germany), as part of a TEXUS module. The thawed spermatozoa were observed under a negative phase contrast microscope (Nikon ELWD 20x objective; Nikon CFW 15x ocular). The samples were inserted into the payload about 40 min before launch. After lift-off, the semen samples in the chamber could be monitored directly online with a TV camera. The experimenter on the ground was able to influence and control experiment parameters via telecommand channels to maintain focusing sharpness. Another channel allowed 12 (TEXUS 19) and 17 (TEXUS 26) different fields of vision during the µg phase. The images were recorded on the ground.
The video tapes were evaluated by a computerised semen motility analyser (Ibac-Cell, AT-CAS 188, Brunner, Munich, Germany), which allowed the measurement of total and progressive sperm motility as well as determination of sperm velocity.2-5 The frame rate for motility analysis was 25/s, the time between successive frames was 40 ms, and the actual observation time for each spermatozoon was 600 ms. Only those cells that could be detected in eight successive frames were evaluated. Curvilinear velocity was calculated from the sum of the straight line distances between all points along the track. Straight line velocity was calculated from the straight line distance between the track's first and last points. In addition, within the group of progressively motile spermatozoa, three classifications could be distinguished, characterising the quality of motion6 by calculation of the linearity values (ratio of straight line to curvilinear, S/V; Fig. 1).
Fig. 1. Examples of motion patterns of spermatozoa. Motility characteristics derived from video acquisitions and processing of the sperm track. V = curvilinear velocity (µm/s); S = straight line velocity (µm/s), showing different motion patterns within progressively motile spermatozoa. Progressively motile spermatozoa are divided into three groups according to their S/V ratios: 0.9-1.0 (linear forward); 0.8-0.9 (not linear forward); 0.0-0.8 (other movement pattern).
The motility of another aliquot of the same ejaculate was examined under identical conditions on the ground to compare with the TEXUS 19 data. For TEXUS 26, the reference test was carried out immediately before launch using the same sample. The advantage of using the same sample was the elimination of variations within the measurements normally found in different aliquots of the same ejaculate.
Totals of 761 and 698 spermatozoa were evaluated under 1 g and 0 g, respectively, on TEXUS 19. By increasing the fields of vision, 1154 and 1090 could be observed during TEXUS 26. The Wilcoxon signed rank test was used for analysis of significance.
In TEXUS 19, no differences between quantitative sperm motility (which refers to velocity) under weightlessness and under gravitational conditions on Earth were detectable. Total motility and the percentage of spermatozoa with progressive motility (velocity >20 µm/s) were nearly identical under 1 g and µg conditions (Fig. 2). However, computer analyses showed significant alterations in motility pattern, i.e. changes in the path shape, expressed as the linearity value. The number of linear forward motile spermatozoa (linearity value >.9) rose significantly (Fig. 3) from 42±9% in the ground reference test to 73±8% in the µg test (p<.005).
Fig. 2. Comparison of quantitative motility on the ground (1 g) and under weightlessness (µg) in TEXUS 19. Spermatozoa are classified into three groups according to their velocity values. a: total motility; all spermatozoa that move >10 µm/s. b: progressive motility; all spermatozoa that move forward at >20 µm/s. c: local motility; all spermatozoa that move at 10-20 µm/s. n.s. = not significant.
Fig. 3. Moving patterns of progessively motile spermatozoa on the ground compared with those under weightlessness in TEXUS 19. The path shape is characterised and expressed by the S/V ratios (= linearity values). These are defined as in Fig. 1.
On the other hand, there were no significant differences in straight line and curvilinear velocities. Velocity values showed only small deviations for both samples under weightlessness and the reference on Earth (Table 1). However, these small differences are sufficient to shift the S/V ratio so that the motion of linearly moving spermatozoa rose significantly. For TEXUS 26, total motility was 71.4±7.2% in the reference test and 79±6.2% under µg conditions (p<.005). A highly significant statistical difference could be observed in the group of progressively motile spermatozoa (Fig. 4). The portion of progressive motility increased from 23.9±9.5% under ground conditions to 49±7.6% (p<.005) during the flight. Curvilinear velocity values within this group also rose significantly from 28.8±2.5 µm/s on Earth to 31.9±3.2 µm/s in space (p<.01; Table 1). At the same time, local motility decreased from 47.5±9.1% in the reference test to 30±6.5% in the µg test (p<.005; Fig. 4). This implies that 17.5% of the local motile spermatozoa (defined as cells with velocities of 10-20 µm/s) could overcome the theoretically defined velocity borderline of 20 µm/s. Under the influence of weightlessness, these cells could be classified by computer analysis as progressively motile spermatozoa. Regarding qualitative motility patterns (path shape), the portion of linear forward motile spermatozoa with linearity values >.9 rose significantly from 30.5±12.8% on the ground to 42.2±16.1% (p<.01) during the flight (Fig. 5). This means an increase in linearity of 12% under the influence of weightlessness.
Table 1. Velocity data of bull spermatozoa on Earth and under weightlessness. S=Straight line velocity (µm/s). V = curvilinear velocity (µm/s). TEXUS 19 TEXUS 26 µg 1 g µg 1 g ------------------------------------------------------------------------------- Velocity (S) 29.1±9.4 27.5±6.2 26.9±2.8 23.8±2.5* Velocity (V) 29.8±9.9 30.9±7.5 31.9±3.2 28.8±2.5** -------------------------------------------------------------------------------- *p<0.005 **p<0.01
Fig. 4. Comparison of quantitative motility on the ground (1 g) and under weightlessness (µg) in TEXUS 26. Spermatozoa are classified into three groups according to their velocity values. a: total motility; all spermatozoa that move >10 µm/s. b: progressive motility; all spermatozoa that move forward at >20 µm/s. c: local motility; all spermatozoa that move at 10-20 µm/s.
Fig. 5. Motion patterns of progessively motile spermatozoa on the ground compared with those under weightlessness in TEXUS 26. The path shape is characterised and expressed by the S/V ratios (= linearity values). These are defined as in Fig. 1. n.s. = not significant.
Using bull spermatozoa, it was possible for the first time to show the influence of gravity on the motility of biological systems. The first indications of µg effects were detectable in the TEXUS 19 experiment, and could be confirmed after reflight on TEXUS 26. The differences in results between the two flights were a consequence of the improved experimental conditions on the second mission. The use of the same ejaculate sample for both the reference and µg tests was highly beneficial. On TEXUS 19 the reference test was carried out by thawing another sample of the same ejaculate.
The detected changes in motility characteristics are definitely due to weightlessness, as the results of the experiments demonstrate. Additional proof was provided by a long series of supporting ground experiments in which launch conditions such as acceleration (12 g) and vibration were simulated. No changes in sperm movement properties were detectable in any case. Microgravity conditions thus favour the motility pattern of spermatozoa and might possibly improve fertilisation capacity. On the other hand, there is still a wide range of explanations for these clear effects.
Possibly the enhancement is due to changes in membrane permeabilities and/or energy balances. It might be that, under µg, energy-supplying mechanisms are more economical and more rapid. The activity of membrane-bound enzymes such as thymidine kinase (E. C. 18.104.22.168) might be influenced by µg-induced changes of the membrane permeability, affecting DNA synthesis.7
Results from other space missions substantiate the above assumptions, proving that weightlessness can have an influence at cellular level. The Franco-Soviet Salyut 6 Cytos experiments on a unicellular organism, the Paramecium, showed a strong stimulation of the cell growth rate.8 An increase in cytoplasmic hydration, a drop in total protein content and a change in the electrolytic content were found, among other effects. This was particularly shown by a decrease in intracellular calcium, probably related to structural changes in the cytoskeleton proteins, especially in their sites for calcium binding, or to modifications of the energetic metabolism connected with ciliary movement.9
Ref. 10 reported that embryonal lung cells (WI-38) cultivated under µg conditions showed a 20% reduction in glucose consumption compared to ground-based experiments. This suggests that energy consumption was lower for cells cultured in the absence of gravity. On the basis of these findings and the results from the TEXUS experiments, it would be of great interest to extend these studies to other biological systems, especially to primate cells.
This work was supported by grants from the German Federal Ministry of Research and Technology (contract number QV 87010). The authors would like to thank Dr O. Haeger, Test and Insemination Station Grub, Germany, for providing bull spermatozoa, and Mrs A. Full for her expert technical assistance.