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Regular Article |
a Department of Molecular & Integrative Physiology,
b Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas 66160
c Microgravity User Support Center, German Aerospace Center (DLR), 51147 Köln, Germany
ABSTRACT
Sperm and other flagellates swim faster in microgravity (µG) than in 1 G, raising the question of whether fertilization is altered under conditions of space travel. Such alterations have implications for reproduction of plant and animal food and for long-term space habitation by man. We previously demonstrated that µG accelerates protein phosphorylation during initiation of sperm motility but delays the sperm response to the egg chemotactic factor, speract. Thus sperm are sensitive to changes in gravitational force. New experiments using the NiZeMi centrifugal microscope examined whether low hypergravity (hyperG) causes effects opposite to µG on sperm motility, signal transduction, and fertilization. Sperm % motility and straight-line velocity were significantly inhibited by as little as 1.3 G. The phosphorylation states of FP130, an axonemal phosphoprotein, and FP160, a cAMP-dependent salt-extractable flagellar protein, both coupled to motility activation, showed a more rapid decline in hyperG. Most critically, hyperG caused a
50% reduction in both the rate of sperm-egg binding and fertilization. The similar extent of inhibition of both fertilization parameters in hyperG suggests that the primary effect is on sperm rather than eggs. These results not only support our earlier µG data demonstrating that sperm are sensitive to small changes in gravitational forces but more importantly now show that this sensitivity affects the ability of sperm to fertilize eggs. Thus, more detailed studies on the impact of space flight on development should include studies of sperm function and fertilization.
fertilization, gamete biology, immunology, signal transduction, sperm, sperm motility and transport
INTRODUCTION
Successful fertilization is critical for reproduction in plants and animals. As plans for extended manned habitation in space proceed, the question of whether space flight conditions have an impact on the ability of species to reproduce [1] has become a current area of emphasis of the NASA program for Gravitational Biology and Ecology. Gravity-sensitive biological processes have been found to be affected by conditions of hypergravity (greater than 1 G) and microgravity (less than 1 G). Changes in the gravitational field been found to have a significant effect on the development of plants and animals [2]. Earlier studies in this and other laboratories demonstrated that conditions of microgravity had significant effects on sperm motility, and we were able to correlate these effects with biochemical changes in the phosphorylation state of axonemal proteins [3]. These studies on sperm motility were limited, however, by the lack of an assay for the functionality of sperm exposed to altered gravity. The objective of the present work has been to test further the hypothesis that alterations of the gravitational field affect sperm motility parameters by exposing sperm to hypergravitational forces. The significance of the gravitational effects was also tested by quantifying the ability of these sperm to bind to and fertilize eggs.
Reduced gravitational field has been shown to have a positive effect on motility of cells that utilize an axonemal apparatus for movement [4, 5]. To date, only one published study has tested the effect of hypergravity on sperm motility [6], and that study used extremely high gravitation forces (1280 x G), which stratified the cell structure. Analyses of the effect of microgravity on fertilization have yielded conflicting results [7, 8]. The sea urchin sperm has provided an excellent model for these studies because of the ease of storage and activation of the sperm [3, 9], however, other species have also been used in microgravity [2, 7, 10, 11] and hypergravity [10, 12–14] experiments to study later aspects of reproduction.
Due to the expense and manpower required to plan, execute, and analyze experiments in µG, it has become a desirable prerequisite to study biological systems in hyperG. Such studies can be carried out in specialized ground-based facilities [15]. If an important biological system is shown to be sensitive to changes in gravitational forces in hyperG and/or simulated µG, then expenditure of the resources needed to test effect of µG by performing an experiment on the Space Shuttle or International Space Station can be more readily justified. With regard to fertilization, hyperG forces up to 3 G are experienced during the first 8 min of launch on the Space Shuttle, and up to 8 G occurs in some turning maneuvers in jet fighter aircraft. Thus, while of minor physiologic significance to fertilization, hyperG can be used to determine whether sperm and sperm-egg interactions are sensitive to gravity as a foundation to examining effects of µG on these processes.
We report here on the development of new hardware and methods that enable us for the first time to study acute gravitational effects on sperm motility, biochemistry, and function. We present data that sperm motility is sensitive to gravity increased as little as 0.3 times above normal gravity. Percent motility (MOT) and straight-line velocity (VSL) were both significantly inhibited, whereas all other motility parameters were stimulated in hyperG relative to 1 G. Two flagellar proteins, FP130 and FP160 (a cAMP-dependent phosphoprotein), showed rapid postactivation declines in phosphorylation in hyperG consistent with the inhibition of MOT and VSL. Because MOT and VSL combine to determine the net forward movement of sperm toward the egg, this suggested that fertilization might be diminished in hyperG. In fact, sperm binding and fertilization were both inhibited by about 50%, suggesting that sperm are more sensitive to changes in gravity than eggs. These results expand previous observations that sperm are sensitive to loss of gravity to include marked sensitivity to increased gravity as well. Thus, plans for extended space flight need to include more detailed studies of the effect of µG and hyperG on sperm activation and fertilization in actual space flight conditions.
MATERIALS AND METHODS
Reagents
All reagents were ACS grade or better unless specified otherwise. Polyclonal rabbit antiphosphothreonine antibodies were obtained from Zymed Laboratories Inc. (San Francisco, CA). L-Polylysine (Sigma P8920) was obtained from Sigma-Aldrich Corp. (St. Louis, MO). Water was deionized using the MilliQUF Plus system (Millipore, Bedford, MA). H89 and H85 were obtained from Seikagaku America (Falmouth, MA). DRB (5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole) and GF 109203X (bisindolylmaleimide I) were obtained from Calbiochem (La Jolla, CA).
Sea Urchin Eggs and Sperm
Sea urchins (Lytechinus pictus or Strongylocentrotus purpuratus) were obtained from Marinus (Long Beach, CA), shipped directly to Germany, and maintained in aquariums at the Microgravity User Support Center (MUSC) facility. Sperm were collected dry for fertilization studies. For motility and protein phosphorylation studies, sperm were collected into sperm storage buffer (MSSB) as described previously [9]. Eggs were collected fresh each day as described by Kinsey et al. [16]. Egg jelly coat was removed by passing the egg suspension through a 150-mesh nylon membrane and washing once with artificial seawater (ASW) at neutral gravity. Eggs and sperm were stored at 12°C prior to use on the same day.
NiZeMi Centrifugal Microscopy
Microscopy was performed under centrifugal force to create hypergravity environments ranging between 1.1 and 5 G. To accomplish this, the NiZeMi slow rotating centrifuge microscope (at the MUSC at DLR in Köln, Germany) was used [15, 17]. The NiZeMi consists of a modified Zeiss microscope and charge-coupled device video camera mounted horizontally on a computer-controlled turntable. For these experiments a 5x phase objective was used. The NiZeMi has a remote-controlled stage, allowing the user to move the stage in the x, y, and z planes using a joystick. The NiZeMi also has remote-controlled servomotors that are used to actuate activation and fixation chambers on special specimen viewing chambers (see below). All controls of the microscope and turntable can be modified while the microscope is rotating.
Two types of observation chambers were used for microscopy. For activated sperm, plastic Petri dishes fitted with 32-µm depth O-rings were used as described previously [18]. For observation of sperm and eggs that could be activated and fixed while under centrifugation, a new activation-fixation observation unit (AFOU) was designed and constructed by the MUSC. The AFOU is made of plexiglas and contains a 2-mm depth optically flat observation chamber with a capacity of 560 µl (Fig. 1). Directly connected to the observation chamber are two rotating discs. One disk contains the activation chamber (120 µl capacity) to hold sperm; the second disk holds fixative (180 µl capacity). Servomotors mounted on the NiZeMi that are controlled remotely by the user separately activate each disk. The rotating edge of the disks is in contact with the fluid in the observation chamber so that when actuated, the rotating disk mixes the contents of the added fluid as it enters the viewing chamber.
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Details of the sequence of centrifugation, activation and fixation are detailed in the Results section for each experiment. For motility analysis, the field of view was changed every 10 sec during a 2-min recording period. Motility was recorded at 1 G for 2 min, prior to activation of the centrifuge, then for 2 min at the test G-force, then for an additional 2 min at 1 G. The test G-forces were preprogrammed into the NiZeMi so that at the end of the prespin 1 G period, hyperG was obtained by throwing a switch on the NiZeMi. Acceleration from 1 G to the test G-force took 2–12 sec, for 1.1–5 G, respectively. Due to a slight change in focus that occurred during the acceleration period, motility was not analyzed during the brief acceleration interval. Video recordings were made by NTSC standard so that the videotapes could be subsequently analyzed in the laboratory at the University of Kansas Medical Center (KUMC).
Sperm Motility Analysis
For analysis of sperm motility, 10 µl of activated sperm was loaded in the petri dish viewing chamber, then sealed with a plastic coverslip. The dish was mounted immediately on the NiZeMi microscope stage. Motility at 1 G, then hyperG, then 1 G was recorded as described above. Sperm motility on the NiZeMi video tapes was analyzed by computer-assisted sperm motility analysis (CASA) using the CellTrack/S system (Motion Analysis Corp., San Francisco, CA) as described previously [3, 9, 19]. All experiments were performed in triplicate. For examination of pelleting effects of hyperG on sperm, the minimum threshold velocity was set to 0 µm/sec for control experiments on fixed sperm. For all other experiments the minimum threshold velocity was 20 µm/sec.
Sperm Activation Protein Phosphorylation Analysis
A 120-µl aliquot of immotile sperm in MSSB [9] was loaded into the AFOU activation chamber. The observation chamber was loaded with 340 µl of hardware sea water (HSW) and 180 µl of 2x electrophoresis sample buffer [20] was loaded into the fixation chamber. The sealed AFOU was mounted onto the NiZeMi and brought to 1 G (no rotation) or 2 G. After 30 sec at the desired G-force, sperm were injected into the observation chamber. Activation was allowed to proceed for 30 or 60 sec and then terminated by actuation of the fixation chamber. Zero-second controls were obtained by injecting sperm into the observation chamber containing a solution consisting of 340 µl HSW that had been premixed with 180 µl of electrophoresis sample buffer. All experiments were repeated in triplicate.
Cyclic AMP-dependency of phosphorylation during activation of sperm was examined as above in microfuge tubes at 1 G rather than using the NiZeMi centrifuge. Sperm were activated as above with HSW containing 20 µM H89 or H85 as a control, then processed for Western analysis of phosphothreonine (pT) as described below [19, 21].
Western Analysis of Protein Phosphorylation
Whole sperm samples fixed for Western immunoblotting as described above were removed from the AFOU, boiled for 10 min, then centrifuged at 200 000 x g for 2 h at 5°C to pellet DNA. Phosphorylation was analyzed as performed previously by Western analysis using anti-phosphothreonine antibodies and ECL (Amersham, Piscataway, NJ) detection of the secondary antibody [3, 19, 21]. Prior to running the Westerns, a separate SDS-PAGE gel was run of each sample and stained with Coomassie blue to verify that each sample had equivalent protein concentrations. To confirm equal sperm protein loading, the Western nitrocellulose sheets, after exposure to the x-ray film, were washed with H2O, then air dried. The sheets were then stained for 60 sec with 0.025% (w:v) Coomassie blue in 40% methanol-1% acetic acid (v:v), fixed with 40% methanol (v:v), washed with H2O, then air dried to visualize the total protein loaded on each lane.
Fertilization Analysis
Fertilization was examined by two methods: sperm-egg binding and elevation of the fertilization envelope [16]. Prior to setting up experiments in the AFOU, fertilization of freshly collected eggs was checked in 96-well microtiter plates to determine the optimal concentration of sperm to inseminate eggs and to verify viability of the eggs (using elevation of the fertilization envelope as a criterion).
The AFOU was loaded as follows: First, a stock 0.1% poly-L-lysine solution was diluted 1:10 000 in H2O and loaded into the observation chamber. After 30 sec, the polylysine solution was removed, and the chamber was rinsed with 5 ml of H2O and then aspirated. Eggs (340 µl as a 1% v:v suspension) were then slowly loaded into the observation chamber using a 10-mm long blunt-ended 18-gauge needle on a 1-ml syringe. The fixation chamber was then loaded with 180 µl of 4.4% glutaraldehyde in ASW. Dry sperm were then freshly diluted 1:500 into ASW and 120 µl was immediately loaded into the activation chamber. The chamber loading ports were then sealed and the AFOU was mounted on the NiZeMi microscope. As soon as the eggs were brought into focus, video recording was started and the NiZeMi centrifuge was brought to the test speed. After 30 sec at the test speed, the sperm activation chamber was actuated. For analysis of sperm-egg binding, the fixation chamber was activated at 20 sec postinsemination. For fertilization envelope elevation, fixation was performed 120 sec postinsemination. Immediately after fixation, the centrifuge was stopped. The AFOU was removed from the NiZeMi, and eggs were analyzed on a Leitz inverted microscope (Wetzlar, Germany) using phase optics. For each of three replicates, 20–25 eggs were counted.
RESULTS
Sperm Motility in Hypergravity
Previous studies have shown that sperm motility [4] and flagellar protein phosphorylation [3] are stimulated in µG. In order to determine whether hyperG had an opposite effect on sperm motility, we measured sperm motility in different G forces between 1.1 and 5 G. These G-forces are 600–800 times lower than those used to centrifuge sperm during normal washing procedures. However, to rule out the possibility that these G-forces might produce motility effects by pelleting the sperm, a control was devised in which sperm were rendered immotile with 0.001% Tween 20 in ASW. Motility was then analyzed at 1, 2, and 5 G in triplicate fixed sperm samples. In order to ensure that centrifugation was not having a pelleting effect on the sperm the minimum threshold velocity for these experiments was set to 0. None of the G-forces were sufficient to produce movement or motility of the sperm in the solution due to the low centrifugal forces (data not shown).
For each test G-force, motility was then examined in live sperm. Prior to application of hyperG, motility was recorded to establish pretreatment values. Motility was measured at hyperG then again at 1 G to determine if hyperG exerts a protracted effect on motility (right bar in each pair, Fig. 2). Each experiment was performed in triplicate. Changes in motility were normalized relative to the value obtained at 1 G prior to the respective hyperG spin for each replicate. Figure 2 shows that percent MOT and VSL were inhibited in hyperG relative to the prespin 1 G controls. There was a similar additional inhibition of both parameters that occurred between hyperG and the postspin recovery period at 1 G. On the other hand, curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), and average path velocity (VAP) were all stimulated when exposed to hyperG but showed no significant change between hyperG and the postspin 1 G period. In other words, the higher VCL, ALH, and VAP motility values obtained in hyperG were maintained in the postspin 1 G period. Thus, it appears that MOT and VSL are negatively impacted by G-forces produced by acceleration and deceleration. On the other hand, VCL, ALH, and VAP are stimulated by hyperG, but this stimulated motility is not reversed when a 1 G environment is restored (within the time frame of these experiments).
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Table 1 presents a summary of the minimum applied G-forces that resulted in a significant change (P < 0.05 or lower) in the motility parameters shown in Figure 2. All parameters that were affected by the increase in G-force required an increase of only 0.3–0.4 G above 1 G. A similar sensitivity was noted for the decline in MOT produced by deceleration from hyperG to 1 G. On the other hand, the reduction in VSL that occurred between hyperG and 1 G required a much larger difference in G-force to see this effect (deceleration from 4 to 1 G).
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Protein Phosphorylation During Sperm Motility Activation in Hypergravity
The previously reported stimulation of sperm motility in µG [4] was matched with an increased rate of phosphorylation of FP130, an axonemal protein modified by threonine phosphorylation [3]. The effect of hyperG on sperm motility parameters described above suggested that FP130 phosphorylation might also be altered in hyperG. To be able to compare the results obtained in µG with results obtained in hyperG it was necessary to develop hardware that would allow sperm to be activated and fixed while being centrifuged on the NiZeMi microscope. The AFOU (see Materials and Methods) filled this requirement. Immotile sperm were maintained at 1 G or brought to 2 G then activated to swim for 0, 30, or 60 sec, then fixed at the same G-force with sample buffer for subsequent Western analysis of phosphothreonine to detect FP130. Figure 3 presents a typical Western analysis showing the region of the gel where FP130 migrates. At 1 G, FP130 showed a time-dependent increase in phosphorylation. However, at 2 G, FP130 phosphorylation increased at 30 sec but declined by 60 sec. Phosphorylation experiments at 5 G were not possible with the current AFOU design because the higher G forces in combination with the presence of SDS in the sample buffer prevented adequate sealing of the chambers and loading ports.
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Gravity-Sensitive 160-kDa Phosphoprotein, FP160, but Not FP130, Is Regulated by cAMP-Dependent Protein Kinase
To determine whether the gravity-sensitive changes in protein phosphorylation involved signal transduction pathways involving cAMP, we examined the effect of H89, a permeable inhibitor of cAMP-dependent protein kinase [22, 23] on phosphorylation of these proteins. As a control, the effect of H85 that has much lower affinity for cAMP-dependent protein kinase [24] was tested. Figure 4 shows that FP160 is inhibited by 20 µM H89 but not 20 µM H85, and that neither inhibitor reduced phosphorylation of FP130. In addition to H89 and H85, permeable inhibitors of protein kinase C (100 µM GF102903X) and casein kinase II (100 µM DRB) had no effect on phosphorylation of either FP130 or FP160 (data not shown).
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Fertilization of Eggs in Hypergravity
The reduction in MOT and VSL, plus the more rapid declines in FP130 and FP160 phosphorylation observed in hyperG suggest that sperm interactions with the egg as well as fertilization may be inhibited in hyperG. This possibility was first examined by quantitation of sperm-egg binding at 1, 2, and 5 G (Fig. 5, left side). Prior to performing these experiments in the AFOU, preliminary bench-top fertilization experiments were conducted in 96-well plates to determine the dose of sperm that gave linear results with respect to time for both sperm-egg binding and fertilization envelope (FE) elevation. Based on these findings, 20 sec was chosen as the endpoint for egg binding and 2 min as the endpoint for FE elevation. It is also important to note that the 20-sec time point was chosen because later time points began to show evidence of FE formation. As FE elevation ensues, many of the bound sperm are released by cortical granule proteases. For all experiments, batches of sperm and eggs that yielded at least 90% FE by 5 min at 1 G were used. Significant reductions in sperm binding measured 20 sec after insemination were observed both at 2 G (P = 0.004) and 5 G (P = 0.04). Fertilization was also examined using elevation of the FE at 2 min postinsemination as a marker (Fig. 5, right side). Significant declines in FE were measured at 2 G (P = 0.003) and at 5 G (P = 0.002) as compared to 1 G values. It should be noted that there was no significant difference in sperm-egg binding or FE between 2 and 5 G. In addition, the extent of the decline in sperm-egg binding and FE from 1 G was about 50% in both cases. This suggests that sperm rather than the eggs are primarily affected by hyperG. If the eggs were also affected by hyperG, then FE formation might have shown a greater decline relative to 1 G than that observed for sperm-egg binding.
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DISCUSSION
A research focus area for NASA concerns whether changes in µG impact the ability of organisms to reproduce [25–31]. Our previous studies demonstrated that protein phosphorylation during activation of sperm motility occurs more rapidly in µG than in 1G [3]. This is consistent with work published earlier by Engelmann et al. [4] showing that sperm swim faster in µG than 1 G. The studies reported here were prompted by our observation that the response to the egg chemotactic peptide, speract, showed a different temporal pattern of protein phosphorylation in µG than in 1 G [3]. While hypergravity is of minor physiologic significance to space flight, nonetheless, it is widely used to explore sensitivity to changes in gravitation that can more easily be tested in the laboratory. Establishment of sensitivity of a biological system to changes in gravitational force (either hyperG or hypogravity, or both) in ground-based laboratory experiments is a critical prerequisite to establish a rationale to examine the impact of µG on such systems. As such, the results reported here further establish the gravisensitivity of sperm and provide key new data suggesting that this sensitivity extends to processes that affect egg fertilization. While the results reported here show that sperm components of fertilization are sensitive to increased gravity, it remains to be determined whether µG also affects early fertilization processes.
A primary target of protein phosphorylation during activation of motility and during the response to speract is FP130. FP130 was previously identified as a tightly bound axonemal protein whose phosphorylation was tightly coupled to activation of sperm motility [19]. FP130 was altered in µG both during activation of motility and also in response to speract [3]. The results reported here extend the µG results and demonstrate that FP130 phosphorylation is also sensitive to hyperG under conditions that change motility as well as sperm-egg interactions and fertilization. Thus, changes in FP130 phosphorylation appear to be very closely coupled to fundamental mechanisms that regulate sperm motility. In addition to FP130, a slightly larger phosphothreonine-containing protein, FP160, also showed parallel changes in phosphorylation in hypergravity. Interestingly, phosphorylation of FP160 was inhibited by H89 but not by H85, suggesting that this phosphoprotein is regulated by a cAMP-dependent protein kinase. In previous studies, FP160 was identified as a sperm phosphoprotein that could be extracted from isolated demembranated flagella by 0.6 M NaCl. Although not discussed in detail in the original paper, examination of the published data show that phosphorylation of this protein also increased during activation of motility [19]. FP160 and FP130 also showed parallel changes in phosphorylation in µG versus 1 G [3]. On the other hand, FP130 was not inhibited by any of the protein kinase inhibitors tested. These results suggest that the gravitational response may involve changes in a balance between activity of both protein kinases and protein phosphatases. Thus, in the case of FP130, the primary mechanism for the decline in phosphorylation may involve activation of dephosphorylation. This is similar to what has been suggested previously not only in the response of FP130 to speract [3] but to other sperm proteins that get dephosphorylated in response to speract as well [25, 26]. Whether or not protein phosphatase activity is altered in the hyperG environment would be difficult to determine directly given the limitations of the currently available hardware for performing these experiments.
The inhibition of sperm-egg binding and fertilization in hyperG raises the question of whether the opposite might be true in µG. With regard to sperm motility and the associated signal transduction, these are both stimulated in µG [3, 4]. Results from Xenopus fertilization suggest that a higher proportion of eggs are fertilized in µG than at 1 G [8]. However, the Xenopus study suggests that even though the differences between µG and 1 G were significant (P < 0.0001), the differences could be due to animal variation. The studies on sea urchins presented here were performed with both the control and hyperG groups that were obtained from the same batch of eggs and sperm. In addition, our sperm studies on the Space Shuttle were performed using ground controls and flight samples that were from the same batch of sperm for each space shuttle mission, respectively [3]. These results in this paper represent the first detailed examination of the effect of hyperG on sperm function and early fertilization processes. Furthermore, the results demonstrate that both µG and hyperG have significant effects on sperm activation, signal transduction, and early fertilization.
It should be noted that our studies are unique in that fertilization was carried out during exposure to hyperG. Nonetheless, it is important to distinguish physiologic consequences of hyperG levels used to wash sperm in the normal laboratory setting from the low hyperG values experienced by astronauts and pilots of advanced performance aircraft. With regard to sperm centrifugation, Brooks [6] found that pelleting centrifugation of bovine sperm produced a dramatic reduction in ATP content and a coupled increase in ADP content. Although the effects were rapidly reversible by resuspension, subsequent exposures to increased gravity produced even greater changes in the content of both nucleotides. There was a gradual but slight decrease in the maximum ATP content after each centrifugation, suggesting damage may have occurred. The G forces that the sperm were exposed to in that study were up to 1280 G, which effectively separated the sperm from glycolysable substrates. In space flight conditions, however, much lower hyperG forces are created (approximately 3 G). As a point of reference, 3 G is experienced during the first 8 min of launch on the Space Shuttle, and up to 8 G occurs in some turning manuevers in jet fighter aircraft. Other key differences that need to be pointed out are that while high-speed centrifugation is normally used to process sperm for fertilization, the changes in sperm motility and ATP content are reversible, and the subsequent fertilization by assisted reproductive technologies is carried out at 1 G. Similarly, we tested hyperG during ground-based control experiments to assess whether Space Shuttle launch conditions of vibration and hyperG affected the subsequent activation of sperm motility [3]. Sperm maintained in preactivation storage conditions during exposure to launch vibration, hyperG, or both showed no difference in subsequent motility when compared to controls. It should, however, be noted that there is one report that whole-body exposure to hyperG in male fighter pilots appears to increase significantly the proportion of female offspring [34]. The mechanism for this effect is unknown.
The results demonstrate that sperm motility comprises different categories of parameters that are distinguished by their sensitivity to gravity. The first group contains parameters that were inhibited by the change from 1 G to hyperG and include MOT and VSL. These parameters were affected not only in the transition between 1 G and hyperG but also continued to decline during the postcentrifugation period after 1 G was restored. The decline in both of these motility parameters may well be an underlying cause of the reduction in sperm-egg binding and subsequent fertilization that was observed in these experiments. A combined decline in the proportion of sperm that are moving as well as the rate at which the sperm are moving toward the egg will decrease the number of sperm encountering the egg. This number is also declining with time under hyperG. Individually, a decline of these two parameters is associated with human infertility [27, 28]. The second group contains motility parameters that were stimulated by the change from 1 G to hyperG (VCL, ALH, and VAP). These three parameters showed no additional change during the postspin 1-G-recovery period but maintained the increased values even after hyperG had been terminated. Even though this last group of parameters was increased in hyperG, the role of these parameters in the net movement of sperm toward the egg may not be of as dramatic consequence in comparison to MOT and VSL. This would be true if sperm-egg collision rate is a factor [37, 38]. If so, then it would be interesting to test whether increasing the sperm concentration would counteract the observed negative gravitation effect. The changes in these motility parameters cannot be attributed to a pelleting effect of centrifugation on the sperm because fixed sperm showed no apparent motility even when the threshold velocity was reduced to 0. Any changes in velocity motility parameters (including VCL, VSL, and VAP) cannot be attributed to inclusion of immotile sperm in the mean of these parameters, as velocity is only measured on sperm swimming at or above the minimum threshold velocity (20 µm/sec).
Regardless of which of the above categories the motility parameters fall into, the effects of altered G-force were retained beyond the brief exposure to hyperG. In this regard, increased viscosity of the fluid medium is known to inhibit sperm motility [29, 30]. We have shown that increased viscosity of the medium, using D2O, inhibits both motility and FP130 phosphorylation [3]. This suggests that changes in protein phosphorylation induced by hyperG could be responsible for the prolonged effect on sperm motility. Further experimentation will be required to determine if motility values return to prehyperG levels with a more extended recovery time at 1 G. Another possible mechanism is that the cytoskeleton detects changes in stress or tension that result in changes in protein phosphorylation. Such a mechanism has been demonstrated in the kinetochore that aids in the correction of misaligned chromosomes during mitosis [31]. In this system, kinetochore protein phosphorylation responds to changes in microtubule tension. In human-osteoblast-like cells, hypergravity induced a marked elevation in phosphorylation of p44/42 MAP kinases [32]. Hypergravity stimulates inositol triphosphate and decreases cAMP levels, resulting in a net increase in phosphorylation of microtubule-associated proteins in HeLa cells [33]. Thus, there is ample supporting evidence from other cell systems to support an effect of gravitational fields on signal transduction resulting in alterations in protein phosphorylation.
There is also a similarity in the effect of changes in gravity on sperm and the well-known gravitropism response in plants. In plants, gravitropism is hypothesized to involve interactions between starch-statoliths, protoplasts with cytoskeletal elements, or both, and signal transduction pathways [34]. Gravitropism in Euglena has been proposed to involve the whole cell body acting as a statolith [35]. In this sense, we propose that sperm would respond to gravity in a similar way. Displacements in the submicrometer range are sufficient to trigger changes in cytoskeletal reorientation [36]. The statoliths and protoplasts are of sufficient mass that the force produced by gravity interacts with cytoskeletal elements, which in turn cause changes in the direction of growth [37–39]. Furthermore, the interactions with the cytoskeleton are coupled to changes in cell signal transduction involving mechanosensitive changes in Ca2+ [5, 40, 41]. A similar relationship between the cytoskeleton and the mass of the sperm head in determining swimming direction in relation to the gravitational vector was proposed by Makler et al. [42]. Similar to plants, Ca2+ is also a key signaling component that regulates changes in sperm movement [43, 44]. Our results support this relationship and suggest that the interactions are sensitive enough to occur under G forces that are below the threshold to cause measurable sedimentation of the sperm during low-speed centrifugation. The sensitivity to small changes in G force could be magnified and extended in time by changes in protein phosphorylation. A gravitation-induced change in sperm Ca2+ could explain the dephosphorylation of FP130 and FP160 that was observed in this study. The presence of protein phosphatase 2B (PP2B), a calcium-calmodulin-dependent protein phosphatase in sperm could regulate these phosphorylations [45, 46]. Although other protein phosphatases such as PP1 or PP2A could also be involved in these dephosphorylations [47–51].
In conclusion, increasing evidence, including new results presented here, suggests that sperm are sensitive to gravitational forces that living organisms, including humans, are exposed to during space flight. Furthermore, it appears that the stimulation of sperm motility and signal transduction during motility activation in µG is reversed in hyperG. In addition, in hyperG the inhibition of sperm motility and protein phosphorylation are associated with significant inhibition of sperm-egg binding and subsequent fertilization. These results suggest that it will be important to assess in greater detail the potential impact of extended exposure to µG on sperm function and fertilization. Although human reproduction in space is not an immediate concern, whether sperm function and fertility are impacted in µG is of importance for the production of food, such as fish, during extended space flight.
ACKNOWLEDGMENTS
The authors acknowledge the untiring support of Stephanie Claassen for assistance in cleaning and reassembly of the AFOU units during our experiments at MUSC, DLR. We thank Dr. Ulrike Friedrich (DLR, Bonn) for her support in providing access to the MUSC and the infrastructure to design, build, and test the AFOU. Critical review of the manuscript by Drs. Ulrike Friedrich and Paul Terranova is gratefully appreciated.
FOOTNOTES
First decision: 23 April 2001.
1 This research was supported by funds from the National Aeronautics and Space Administration (NASA, NAG 2-1016) and by NICHD/National Institutes of Health through cooperative agreement U54 HD-33994 as part of the Specialized Cooperative Centers Program in Reproductive Research. 
2 Correspondence: Joseph S. Tash, Dept. Molecular & Integrative Physiology, University of Kansas Medical Center, Lied G-005, 3901 Rainbow Blvd., Kansas City, KS 66160-7401. FAX: 913 588 7180; jtash{at}kumc.edu
Accepted: May 21, 2001.
Received: April 11, 2001.
REFERENCES
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