(The FASEB Journal. 1999;13:S43-S54.)
© 1999 FASEB
Microgravity alters protein phosphorylation changes during initiation of sea urchin sperm motility
JOSEPH S. TASH1 and
GERÁCIMO E. BRACHO
Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160-7401, USA
1Correspondence: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7401, USA. E-mail: jtash{at}kumc.edu
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ABSTRACT
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European Space Agency (ESA) studies demonstrated that bull sperm swim
with higher velocity in microgravity (µG) than at 1 G. Coupling
between protein phosphorylation and sperm motility during activation in
µG and at 1 G was examined in the ESA Biorack on two space shuttle
missions. Immotile sperm were activated to swim (86–90% motility) at
launch +20 h by dilution into artificial seawater (ASW). Parallel
ground controls were performed 2 h after the flight experiment.
Activation after 0, 30, and 60 s was terminated with
electrophoresis sample buffer and samples analyzed for phosphoamino
acids by Western blotting. Phosphorylation of a 130-kDa
phosphothreonine-containing protein (FP130) occurred three to four
times faster in µG than at 1 G. A 32-kDa phosphoserine-containing
protein was significantly stimulated at 30 s but returned to 1 G
control levels at 60 s. The rate of FP130 phosphorylation in µG
was attenuated by D2O, suggesting that changes in water
properties participate in altering signal transduction. Changes in
FP130 phosphorylation triggered by the egg peptide speract were delayed
in µG. These results demonstrate that previously observed effects of
µG on sperm motility are coupled to changes in phosphorylation of
specific flagellar proteins and that early events of sperm activation
and fertilization are altered in µG.—Tash, J. S., Bracho,
G. E. Microgravity alters protein phosphorylation changes during
initiation of sea urchin sperm motility.
Key Words: space flight • phosphoamino acids • Western immunoblot
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INTRODUCTION
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PLANNING FOR LONG-TERM SPACEFLIGHT and habitation in
microgravity
(µG)2raises the question of whether processes related to sperm motility and
fertilization that occur at Earth-normal gravity (1 G) are altered
under spaceflight conditions of µG (1-4)
. Most
published studies concerning fertilization in µG have focused on
post-fertilization embryo development and not sperm or sperm-egg
interactions per se. Investigations on sperm motility and
spaceflight have been limited to one study (5)
. In this
European Space Agency (ESA) experiment, samples of bull sperm in semen
showed a significant increase in progressive motility and mean
curvilinear velocity, whereas straight line velocity was decreased
significantly. In contrast, studies on fertilization of
Xenopus eggs in µG found that cortical contraction in
response to sperm penetration occurred more rapidly than at 1 G
(6)
. This suggests that either the sperm were able to
fertilize the eggs more rapidly and/or the initial events triggered by
fertilization of the egg occur more rapidly in µG. It should be noted
that both ESA studies utilized sounding rockets, which exposed the
sperm and eggs to launch vibration and acceleration much higher than
those experienced during space shuttle launches.
The activation of sperm motility and subsequent alterations associated
with capacitation in mammalian sperm or exposure to egg peptides in
echinoderms are known to be regulated by changes in protein
phosphorylation and levels of calcium (Ca2+) and cAMP
(mammalian) or cAMP and cGMP (echinoderms) (7-11)
. We
have developed a new method for spawning and storing live immotile sea
urchin sperm (12)
. Using this new model, we have examined
the protein phosphorylation changes that occur during the initiation of
motility in live sperm (13)
. We found that phosphorylation
associated with the de novo activation of motility is mainly
limited to flagellar serine-containing proteins of 32 and 29 kDa and
threonine-containing proteins of 130 and 500 kDa. In addition, we found
that the level of protein phosphorylation before activation of motility
is extremely low and limited to proteins in the sperm head. The 130-kDa
protein, termed FP130, has become a prime focus of research in our
laboratory. In mouse sperm, a related protein of 120 kDa has identical
motility-coupled phosphorylation properties, and a major 120-kDa
threonine-containing protein is also present in live swimming human
sperm (14)
. We also observed that environmental toxins
that inhibited sperm motility caused FP130 dephosphorylation (Tash,
Bracho, Szabo, and Rozman, unpublished observations).
The results of two experiments conducted in the ESA Biorack aboard the
orbiter Atlantis on separate NASA space shuttle missions to
the Russian Space Station MIR are reported. Ground controls
were performed in identical Biorack hardware using aliquots of the same
sperm suspension used in flight. We report here that µG has a
significant stimulatory effect on the rate of protein phosphorylation
during initiation of sperm motility when compared to sperm activated at
1 G. This stimulation occurred in sperm from both Lytechinus
pictus and Stronglyocentrotus purpuratus. In addition,
the proteins affected in µG appeared to be the same flagellar
proteins previously identified as putative subunits of dynein. We found
that the chemotactic egg peptide speract also targets FP130, and that
its time-dependent phosphorylation was different in µG. Finally,
heavy water attenuated sperm motility and the rate of FP130
phosphorylation during activation, suggesting that changes in water
properties may contribute to alterations in signal transduction in
µG. We conclude that published data demonstrating stimulation of
sperm motility in µG is supported by an increase in the
phosphorylation of flagellar proteins. In particular, FP130 appears to
be intimately involved in regulation of sperm movement.
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METHODS
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Materials
Sea urchins (Stronglyocentrotus purpuratus and
Lytechinus pictus) were obtained from Marinus (Long Beach,
CA). Polyclonal anti-phosphoserine (anti-pS) and anti-phosphothreonine
(anti-pT) and monoclonal anti-phosphotyrosine (anti-pY) antibodies (Ab)
were from Zymed Corporation (San Francisco, CA). Peroxidase-conjugated
secondary Ab was from Pierce (Rockford, IL). Chemiluminescence reagents
for Western analysis were from Amersham Corp. (Arlington Heights, IL).
All other reagents were analytical grade.
Buffers
The following buffers were routinely used: artificial
seawater pH 8.0 (ASW), sperm activating buffer (HSW) was ASW adjusted
to pH 8.3 (HSW was prepared without KCl to allow for the dilution with
MSSB, which provides sufficient KCl for the final activated mixture);
MES sperm storage buffer (MSSB), consisted of ASW except that it
contained 50 mM KCl and 5 mM MES and was adjusted to pH 6.0. The
composition of these buffers was described previously (12
,
13
). For heavy water experiments, buffer compositions were
identical to those described above, except that 100% D2O
was used instead of H2O. Buffers made with D2O
were designated with the prefix heavy, i.e. ASW (H2O)
versus heavy ASW (D2O).
Preparation of sperm
For µG experiments and concurrent ground controls, animals
were housed in laboratory facilities at Kennedy Space Center. Immotile
sperm were prepared essentially as previously described
(12)
. The two space shuttle experiments were performed on
STS-81 (January, 1997) and on STS-84 (May, 1997). These dates
necessitated the use of S. purpuratus on STS-81 and L.
pictus on STS-84 due to the seasonal breeding of these species.
Previous studies demonstrated that the phosphoproteins coupled to
activation of motility in these species were identical
(13)
. A pool of sperm from several animals was made to
minimize individual variations. Sperm in MSSB were always adjusted to a
final concentration of 1 x 108 cells/ml before
loading into the hardware. Storage of sperm was always at 5°C.
For heavy water experiments, after rinsing the animals in MSSB, 1 ml of
heavy MSSB was dropped over the spawning ducts of the animals to
replace the H2O with D2O on the surface of the
animals. The animals spawned for heavy MSSB were then collected dry and
diluted into heavy MSSB to adjust the final sperm concentration to be
identical to the sperm in normal MSSB.
Flight and ground control hardware
The Biorack has been employed successfully in five prior space
shuttle missions (15-17)
on STS-42 (January, 1992),
STS-61A (October, 1985), STS-65 (July, 1994), and STS-76 (March, 1996).
ESA provided the Phorbol hardware (Fig. 1
), manufactured by CNES/COMAT, that was utilized for these
experiments. This hardware had been used previously on STS-65 and
STS-76. Each cassette contains six independent sets of three chambers
(Fig. 2
) comprising a sperm chamber, a culture chamber, and a fixation
chamber. The sperm chamber and fixation chambers can each be connected
to the culture chamber by turning sluices that open independent
channels between them. After opening the sperm sluice, a six-pronged
activation tool simultaneously presses the plungers of the six sperm
chambers, thus injecting the sperm into the six culture chambers
containing HSW. The sperm sluice is then closed and the fixation sluice
is opened. After 30 or 60 s of activation, the fixation plungers
are then pressed, thereby injecting the fixative into the culture
chamber, thus terminating motility and phosphorylation.
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Figure 1. ESA bioreactor cassettes, Phorbol, used in the Biorack for sperm
activation and fixation. The type I container (A) is used to
seal and hold the cassette in containment racks and provides a level of
containment during storage. The hardware cassettes (B) hold
six independent sets of three interconnecting chambers. Each chamber
set comprises an activation chamber, tissue culture chamber, and
fixative chamber. Six units were flown on Shuttle flight STS-81 and
twelve units on STS-84. An exploded diagram describing the loading of
the cassettes is presented in Figure 2
.
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Figure 2. Starting configuration of Phorbol hardware. This diagram details the
interconnecting chambers of the Phorbol hardware depicted in Figure 1
and how it was loaded. Details of the solutions are presented in
Methods. Each cassette was used for a single time point (0-, 30-, or
60-s activation). In the control cassettes (0 s), the culture chamber
contained HSW premixed with sample buffer.
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Pre-flight hardware loading and stowage
For each space shuttle flight, three replicate sets of
cassettes were prepared. One set was prepared for flight on the orbiter
Atlantis (OV-104), one set for the ground control, and a
third set was prepared as a backup in case of a launch scrub. Each set
was comprised of two cassettes for each 0-, 30-, and 60-s time point,
respectively, as detailed in Table 1
. All flight hardware was to be handed over 18 h before launch
in order to allow sufficient time for the hardware to be stowed in the
Orbiter. Thus, sperm were spawned into MSSB 24 h before handover
as described above. After adjustment of the sperm concentration, 80
µl of sperm in MSSB were loaded into each of the six sperm chambers
in pre-cooled (5°C) cassettes. Before loading the sperm, the hardware
had already been loaded with 340 µl of HSW and 150 µl of 4x sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer (18)
in each of the six culture and fixative
chambers, respectively. The cassettes to be used for 0 s were
loaded with HSW premixed with 4x SDS-PAGE sample buffer (total volume
of 490 µl) to ensure that the sperm would be fixed at a true 0-s time
point. The sperm chambers were loaded with a small air bubble within
the chamber to facilitate complete injection of the sperm sample into
the HSW and to promote mixing during experiment activation. After leak
tests, cassettes were loaded into type 1 containers, placed into PTCUs,
and then loaded into the orbiter Atlantis. Cassettes were
oriented so that during launch, the gravitational vector was toward the
sluice between the sperm chamber and the culture chamber.
Sperm activation and fixation
The ground control sets were handled using the same time line as
the flight set, except that the 1 G MET clock was started 2 h
later than the space shuttle launch time. This delay allowed time for
anomalies that might occur in the flight experiment to be incorporated
into the ground controls so that the only variable would be µG. The
flight experiment was monitored on the ground with live video downlink
of the Biorack glovebox manipulations as well as air-to-ground audio
from the orbiter to Johnson Space Center (JSC) and the Experiment
Monitoring Area (EMA) at Kennedy Space Center (KSC). Identical
cassettes with replicate Biorack equipment and PTCUs at KSC were used
for the 1 G controls 2 h later. No anomalies occurred during the
STS-81 and STS-84 SPERM experiments, thus the third set was used as a
replicate 1 G control. At approximately 19 h MET, the cassettes
were removed from the 5°C PTCU and allowed to equilibrate to 22°C.
After 50 min, each cassette was processed for motility activation,
followed by fixation at 0, 30, or 60 s, and finally placed in the
onboard -20°C freezer for the remainder of the 10-day mission.
Processed ground controls were also placed in a -20°C freezer for
the same period of time. After shuttle landing at KSC, all flight and
ground cassettes were removed from freezers, kept frozen, and shipped
to the laboratory at University of Kansas Medical Center, where they
were maintained at -20°C until preparation for Western analysis.
Ground controls for launch vibration and acceleration
Due to the short activation time for these experiments, the 1 G
centrifuge on the Biorak was not usable. To account for possible
effects of launch vibration and acceleration on the sperm, hardware
loaded with immotile sperm in MSSB were subjected to a simulated
shuttle launch. Acceleration G-force profile was achieved using the
computer-controlled 40-foot centrifuge, and vibration using the
computer-controlled electromagnetic vibration table at NASA Ames
Research Center (San Jose, CA). The vibration profile was the same as
launch except in one directional vector, instead of three simultaneous
vectors experienced during a real launch. Four sets of hardware were
prepared for these tests: 1 set experienced both vibration and
acceleration, 1 set experienced only vibration, 1 set only
acceleration, and the last set was transported in parallel with the
other three sets but not exposed to vibration or acceleration. As soon
as each test was completed, viability of the sperm was assessed by
injecting the sperm into the culture chamber containing HSW to activate
motility as described above. The activated sperm were then removed from
the culture chamber and motility was recorded immediately by video
microscopy and analyzed by digital image analysis as described below.
Electrophoresis and Western analysis with anti-phosphoamino acid
antibodies
Cassettes were warmed to ambient temperature and samples removed
and centrifuged at 200,000 g for 2 h at 5°C to pellet
the DNA. The supernates were removed, aliquoted, and kept frozen at
-80°C until used for electrophoresis. Western blotting
(19)
detection of pS, pT, and pY was performed on
nitrocellulose membranes containing proteins separated by 5–15%
gradient SDS-PAGE (18)
as described previously
(13)
. Immunocomplexes were detected after 1-min reaction
with ECL reagent and X-ray film exposure for 10–90 s. Complete
activation time courses (0, 30, and 60 s) from µG and 1 G
controls were always run on the same gel to eliminate variability
between gels and ECL reactions. Competitive Western control experiments
were performed as described previously (13)
.
Western signals were quantitated by digital image analysis using GelPro
(Version 3, Media Cybernetics). Gray levels were calibrated to optical
density with the use of a Kodak gray level step tablet. Integrated
optical density (IOD) for each band was corrected against local
background IOD and also adjusted for protein loading in each lane by
staining with Coomassie blue the nitrocellulose sheet used for Western
analysis. The Coomassie blue signal for each lane was compared to lane
1 to obtain a load factor used to adjust the phosphoamino acid IOD
signal in that lane. To compare Western signals from replicate gels,
the IOD values were normalized (IODnorm) by subtracting the
lowest IOD value (IODmin) from each band value
(IODraw) and dividing it by the highest IOD value
(IODmax) for the experiment by use of the following
equation: IODnorm =
(IODraw-IODmin)/(IODmax-IODmin).
Motility analysis
No microscope was available aboard the orbiter to assess
motility of the sperm. To assess the effect of storage on the sperm,
replicate samples of the same sperm were stored at 5°C, warmed to
22°C, then analyzed at KSC for motility activation using the same
flight time line in parallel with the flight experiment. Sperm motility
was videotaped and then analyzed by computer-assisted sperm analysis
(CASA) as described previously (12)
. Video microscopy at
KSC was obtained at 22°C using an Olympus inverted microscope with a
Nikon 20x BM objective and recorded as described above. An identical
microscopy system was used at NASA Ames to record motility in sperm
activated after exposure to launch vibration and acceleration
parameters. Videotapes were analyzed with the CellTrack/s (Motion
Analysis Corp.) system in the laboratories at the University of Kansas
Medical Center.
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RESULTS
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Vibration and acceleration controls
Launching of the space shuttle dramatically increases gravity and
vibration before achieving orbit. High-frequency vibration has been
shown to produce sperm damage, causing leakage of nucleotides critical
to the maintenance of motility (20)
. Centrifugation in
high G forces also significantly alters the ATP/ADP ratio and motility
of sperm (21)
. Even though the sperm were inactive during
this phase, it was necessary to determine whether the vibration or
acceleration experienced during launch might have an effect on motility
initiation. Analysis of sperm motility has long been an accepted means
for assessing damage to sperm (20
, 22
). To
address this question, immotile sperm in MSSB were subjected to
simulated launch vibration, acceleration, or both, and activated by
dilution into HSW. Motility was then analyzed and compared to parallel
controls not exposed to vibration or acceleration. There was no
significant difference between motility of controls versus sperm
treated by acceleration alone, vibration alone, or both acceleration
and vibration (Table 2
). Because no changes in sperm motility were observed,
phosphorylation changes were not analyzed.
Viability of sperm samples during flight
Sperm activation experiments on board the space shuttle were
paralleled with simultaneous experiments conducted on replicate samples
of the same sperm suspension on the ground. As a control, sperm were
diluted into the same volume of MSSB to determine whether the sperm had
remained immotile during the 40-h pre-activation period. Figure 3
summarizes the motility results from both STS-81 and STS-84
experiments. Before dilution into HSW, both percent motility (MOT) and
progressiveness (PRG) of the sperm was extremely low (MSSB). However,
sperm activation significantly increased MOT and PRG. The average
curvilinear velocity of the activated sperm for both experiments was
241 ± 8 µm/s (SEM).
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Figure 3. Motility determination of replicate sperm samples analyzed at 1 G.
Sperm were collected and maintained immotile during loading into the
Phorbol cassettes. At approximately MET 0:21:00, while the flight
experiment was being conducted, replicate aliquots of the same sperm
suspension were activated at KSC using the same flight procedures.
Motility was recorded by video microscopy and quantitated by automated
digital analysis. The left bar in each pair represents the motility
parameter in MSSB before activation and the right bar after
activation by injection into HSW. MOT, % motility; PRG, progressiveness (100 x MOT x curvilinear velocity).
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Phosphothreonine changes during motility activation in 1 G versus
µG
Western analysis of phosphothreonine-containing proteins during
initiation of sperm motility in 1 G versus µG is presented in
Figure 4
.The figure presents a typical Western analysis where a complete time
course of both µG and 1 G samples are run on the same membrane. Sperm
flagella contain a 130-kDa protein (FP130) that becomes phosphorylated
on threonine residues during initiation of motility (13)
.
As presented in Figure 4
, FP130 (indicated by the arrow) was observed
in sperm both in µG and at 1 G. However, there was a dramatic
increase in the temporal pattern of phosphorylation of this protein in
µG, as will be discussed below. In addition to the FP130, the same
set of heavily phosphorylated low-Mr proteins
between 14 and 9 kDa that do not change with motility were observed
both in µG and at 1 G (Fig. 4)
. These low-Mr
proteins were localized to the sperm head, whereas FP130 was localized
to the axoneme (13)
. Based on load-corrected quantitation
of replicate Western blots, there was no effect of µG on the
phosphorylation state of these head-localized phosphoproteins. The data
presented in the next two sections will describe pS and pY changes. The
most dramatic changes in phosphorylation occur with FP130; therefore
the major focus of the report will be on FP130.
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Figure 4. Western analysis of pT in µG (left three lanes) and at 1 G (right
three lanes) in L. pictus sperm. Activation times for sperm
motility are presented below each lane. The increase in phosphorylation
of FP130 occurred faster in µG than at 1 G. Similar results were
obtained in replicate chambers and replicate cassettes. This figure
depicts a typical result obtained when complete time-courses were run
on a single gel. Similar results were obtained in replicate chambers
and replicate cassettes. Data for S. purpuratus (STS-81) and
L. pictus (STS-84) were comparable and showed a similar
time-dependent pattern. See Figure 6
for quantitative statistical
analysis of the changes in phosphorylation of this protein in replicate
samples.
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Phosphoserine changes during motility activation in 1 G versus µG
In addition to the phosphothreonine-containing proteins noted in
Figure 4
, above, previous results have demonstrated that a 32- and
29-kDa pair of proteins are phosphorylated on serine during initiation
of motility (13)
. These same proteins were identified in
sperm activated in both µG and at 1 G, and are indicated in
Figure 5
.Comparison of replicate Westerns (at least six) suggests that the
phosphorylation of these proteins appears to occur more rapidly in µG
than in 1 G. It appears that the magnitude in change of the
phosphoserine proteins (Fig. 5)
is not as great as the change observed
in the phosphothreonine-containing protein (Fig. 4)
.
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Figure 5. Western analysis of pS in µG (left three lanes) and at 1 G
(right three lanes) in L. pictus sperm. Activation times for
sperm motility are presented below each lane. The positions of the
motility-related 32- and 29-kDa phosphoproteins are indicated. The
low-molecular-weight sperm head phosphoproteins at 14 and 9 kDa are
also indicated. An increase in phosphorylation of the 29- and 32-kDa
proteins occurred faster in µG (30 s) than at 1 G (60 s). This figure
depicts a typical result obtained when complete time-courses were run
on a single gel. Similar results were obtained in replicate chambers
and replicate cassettes. Data for S. purpuratus (STS-81) and
L. pictus (STS-84) were comparable. Quantitative statistical
analyses of the changes in phosphorylation of the two bands in
replicate samples are presented in Figure 6
.
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Phosphotyrosine during motility activation in sperm
We have shown previously that phosphotyrosine levels are extremely
low in immotile sea urchin sperm and that there are no significant
changes that occur within the first 60 s of sperm activation
(13)
. Similar results were obtained in these experiments,
therefore the data are not presented.
Quantitative changes in phosphorylation during sperm activation in
µG versus 1 G
Quantitation of Western analyses of the proteins identified in
Figures 4 and 5
from replicate time courses is presented in
Figure 6
.The data were pooled from replicate time-courses from separate
cassettes flown on STS-81 and STS-84. This was possible because the
phosphoproteins in sperm from both species show identical patterns
(13)
. For FP130 (Fig. 4)
, comparison of µG versus 1 G
phosphorylation at each time point by t test demonstrated a
significant effect of gravity at both the 30- and 60-s time points,
respectively (Fig. 6
, top panel). Statistical analysis of the entire
data set by two-way analysis of variance (ANOVA) revealed that the
effect of µG on time-dependent phosphorylation of the 130-kDa protein
was highly significant (P =0.009). It should be noted
that the increase in phosphorylation of FP130 at 1 G was relatively
slower than previously reported (13)
. This slower increase
in phosphorylation is due to the slower rate of motility acquisition
that occurs as the storage time in MSSB increases. In the experiments
reported here, the sperm in µG and at 1 G were maintained in MSSB for
the same period of time before activation, thus the observed
differences in protein phosphorylation relative to µG and 1 G are due
to effects of µG and not storage time.
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Figure 6. Quantitative analysis of protein phosphorylation changes in µG and at
1 G. The phosphothreonine (top panel) and phosphoserine bands (center
and bottom panels), identified in Figures 4 and 5
, were quantitated
using digital image analysis as described in Methods. Error bars
represent the standard error of the load-corrected mean for six
separate sets of chambers (a chamber set being 0-, 30-, and 60-s
chambers from the same experiment run on the same gel) randomly
selected from different cassettes from either STS-81 or STS-84.
P values indicate significantly different values between
µG and 1 G for that time point. Two-way ANOVA analysis of the data is
presented in the text.
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Quantitative analysis of the time-dependent phosphorylation of the 32-
and 29-kDa proteins is presented in Figure 6
(middle and bottom panel,
respectively). Replicate samples were quantitated by digital image
analysis as described in Methods. The 32-kDa protein was phosphorylated
significantly more rapidly in µG than at 1 G (Fig. 6
, middle panel).
Comparison of the µG versus 1 G phosphorylation at each time point by
t test demonstrated a significant effect of gravity at the
30-s time point. Statistical analysis of the entire data set by two-way
ANOVA revealed that the effect of µG on time-dependent
phosphorylation of the 32-kDa protein was highly significant
(P =0.002), whereas the 29-kDa protein was
phosphorylated at similar rates in µG and at 1 G (Fig. 6
, bottom
panel). It should be noted that the 32-kDa protein not only was
phosphorylated more rapidly in µG, but it also showed a subsequent
decline that was not observed in the 1 G control. The decline in
phosphorylation after reaching an initial peak of phosphorylation can
also be observed at 1 G. However, this decline occurs later (about
120 s) in sperm that have been stored for 40 h before
motility activation, as was required by the timeline of the shuttle
experiments (data not shown).
Signal transduction in response to speract is altered in µG
To examine whether early fertilization-related processes are
altered in microgravity, we tested whether the response of sperm to
speract was altered in µG. Speract is a chemotactic peptide released
from the egg jelly coat that binds to receptors on the sperm, causing
the sperm to swim toward the egg. This process causes an initial
increase in protein kinase activity followed by an increase in protein
phosphatase activity (11
, 23
). Immotile sperm
were activated into HSW in the absence (control) or presence of 50 nM
speract in 1 G or µG (Fig. 7
).Quantitative analysis of replicate Western blots is presented in
Figure 8
.As indicated in both the Western photograph and the accompanying
quantitative data, speract caused a dramatic and statistically
significant change in the temporal pattern of FP130 phosphorylation in
both 1 G (Fig. 7
, top panel) and µG (Fig. 7
, bottom panel). In µG,
speract attenuated the rate of increase in FP130 phosphorylation,
whereas at 1 G, there was an initial increase followed by a decrease in
FP130 phosphorylation. The data presented here are the first
demonstration of an effect of speract on phosphorylation of an axonemal
protein. The biphasic pattern of FP130 phosphorylation at 1 G is
similar to that observed for sperm membrane proteins (23)
.
However, the delayed increase in FP130 phosphorylation observed in µG
suggests that a change in the relative timing of changes in protein
kinase and phosphatase signaling has occurred.
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Figure 7. Effect of speract on FP130 phosphorylation. Sperm were activated for 0,
30, and 60 s in ASW (control) or ASW containing 50 nM speract
(speract) at 1 G (top panel) and in µG (bottom panel), and samples
analyzed for pT as described in Methods. Only the region where FP130
migrates (indicated by the arrow) is shown in each panel. The apparent
difference in the 0-s control at 1 G and in µG in the absence of
speract is due to different lane loadings. When adjusted for protein
loading (see Fig. 8
), there was no difference in the 0-s levels of
phosphorylation in the samples.
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Heavy water attenuates the increased rate of FP130 phosphorylation
in µG and 1 G
Heavy water was used as a model to test whether changes in the
density or viscoelastic properties of water may contribute to the
increase in sperm motility and protein phosphorylation in µG. Sperm
were spawned into MSSB made with H2O or D2O,
then activated by dilution into HSW made with H2O or
D2O, respectively. Motility analysis of ground controls
(Fig. 9
)shows that D2O significantly inhibited VCL, LIN, and PRG
during activation of motility. Western immunoblots of FP130 (Fig. 10
)and corresponding quantitation of replicate Western blots (Fig. 11
)demonstrate that heavy water attenuated the rate of increase in FP130
phosphorylation in both 1 G (Fig. 11
, left 3 lanes) and µG (Fig. 11
,
right 3 lanes). Analysis of the time-dependent changes in replicate
Western blots by ANOVA (Fig. 11)
demonstrated that D2O
significantly inhibited FP130 phosphorylation in both 1 G and µG. It
should be noted that the overall time-dependent pattern of inhibition
by D2O in µG versus 1 G was quite different. This could
be due to the fact that in 1 G, there was a gravitational vector as
well as the D2O to alter the environment, whereas in µG,
only the presence of D2O was an environmental variable.
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Figure 9. Heavy water inhibits activation of sperm motility at 1 G. Ground
controls for experiments flown on STS-84 examined whether heavy water
would inhibit activation of motility. Lack of storage space prevented a
microscope being included to examine motility in µG. Sperm were
collected in MSSB made with normal water (H2O) or heavy
water (D2O), then activated in HSW made with
H2O or D2O, respectively. Motility was analyzed
in triplicate as described in Methods. Parameters that were
significantly different from controls (P > 0.001) are
indicated by an asterisk.
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Figure 10. Heavy water attenuates FP130 phosphorylation in µG. Sperm were
prepared in MSSB made with H2O or D2O and
loaded into matched sets of flight and ground hardware as described in
Table 2
. Sperm were activated in µG (left three lanes) or at 1 G
(right three lanes) for 0, 30, and 60 s by dilution into HSW made
with H2O (top panel) or D2O (bottom panel),
respectively. Samples were analyzed for FP130 phosphorylation by
Western analysis of pT. The panels show the region of the gel where
FP130 migrates.
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Figure 11. Quantitative analysis of FP130 phosphorylation in normal and heavy
water at 1 G and µG. Replicate samples of the heavy water experiment
similar to that shown in Figure 10
were analyzed by GelPro. The top
panel compares FP130 phosphorylation during the first 60 s of
activation in H2O vs. D2O at 1 G. The bottom
panel summarizes the load-adjusted data for the same comparison in
µG. Error bars represent SEM.
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DISCUSSION
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|---|
The results presented here demonstrate that µG has a significant
stimulatory effect on protein phosphorylation during initiation of
sperm motility. The primary target for protein phosphorylation changes
in µG is FP130. FP130 has been previously identified as the major
pT-containing protein, tightly bound to axonemes, that is coupled to
initiation of flagellar motility (13)
. A key aspect of the
studies presented here is that the results were repeatable on separate
space shuttle flights using two different species of sea urchins. We
have previously shown that the activation of motility in the buffers
developed for the project and the phosphorylation pattern coupled to
initiation of sperm motility in the two species is identical
(12
, 13
). A stimulatory effect of µG on
protein phosphorylation is consistent with previous observations
demonstrating a stimulatory effect of µG on sperm motility
(5)
. Taken together, these results support the underlying
hypothesis that protein phosphorylation and sperm motility activation
are tightly coupled (13
, 24
).
The targets in sperm affected by µG appeared to be the same
flagellar phosphoproteins shown to be key components of the signaling
pathways that determine initiation of motility (13)
. These
targets include a pair of 32- and 29-kDa phosphoserine-containing
proteins as well as FP130, a phosphothreonine-containing protein.
Although all three of these proteins were found to change during
initiation of sperm motility in both µG and 1 G, stimulatory effects
of µG were observed on FP130 and the 32-kDa protein, but not on the
29-kDa protein. In addition, the magnitude of the µG effect was most
pronounced on FP130. In a previous study, we speculated that FP130 may
be a subunit of inner arm dynein (13)
homologous to the
138-kDa subunit of inner arm dynein 1 identified in
Chlamydomonas flagella (25)
. If FP130 is in
fact an inner arm dynein 1 subunit, then our results indicate that one
of the primary determinants of flagellar motility is a prime target for
the effects of µG in sperm motility. This conclusion is based on the
suggestion that inner arm dynein 1 is the major regulator of flagellar
motility and because double mutants of inner arm 1 and inner arm 3 in
Chlamydomonas are completely immotile (26)
. In
this connection a similar motility-coupled protein has been identified
in mouse sperm (14)
.
This study is also the first to identify FP130 as a target for signal
transduction in response to the chemotactic egg peptide speract.
Binding of speract to receptors on the sperm surface causes initial
increased phosphorylation of membrane proteins and activation of
guanylyl cyclase (11)
. This increase is followed rapidly
by decreased phosphorylation of sperm membrane proteins and a decline
in speract-activatable guanylyl cyclase activity (23)
.
This decrease in membrane phosphorylation was postulated to be due to
enhanced protein phosphatase activity. The data presented here show
that the increase in FP130 phosphorylation during sperm activation is
attenuated by speract and that the temporal pattern of FP130
phosphorylation is different at 1 G and in µG. At 1 G, FP130 showed a
bell-shaped change in phosphorylation similar to the pattern noted for
the membrane proteins in response to speract (11
,
23
). However, in µG, FP130 showed only a delayed
increase in phosphorylation during the 60-s activation period, without
the subsequent decline. This suggests that the timing of activation of
protein kinases and protein phosphatases is altered in µG. The major
effect of speract on sperm motility is to convert the swimming pattern
from predominantly circular paths to straight paths toward the site of
speract release (i.e., the egg), which requires a modification in the
bending pattern of the flagellum. The fact that the FP130 is bound to
the flagellar axoneme suggests that FP130 contributes to the change in
motility triggered by speract. With regard to the fertilization
process, these results raise the question of whether early
fertilization events involving interactions between the sperm and egg
are altered to an extent that the timing and efficiency of
fertilization are affected.
The relationship between gravitational forces and sperm function has
been the focus of several studies. With regard to acute effects of
µG, bovine sperm demonstrated significant increases in curvilinear
velocity and progressiveness. In addition, µG significantly reduced
the proportion of motile sperm swimming at low velocities between 10
and 20 µm/s. These findings suggest that µG alleviates restrictive
influences on the expression of motility. Whether biomechanical changes
in flagellar function are also due to µG-induced changes in the
viscoelastic properties of the fluid in which the sperm swim is another
possibility. We found that heavy water, which has 11% higher viscosity
and density than H2O, inhibits protein phosphorylation and
motility both in µG and at 1 G. Interpretation of the D2O
results is complicated by the fact that at 1 G, the gravitational
vector was present in addition to the heavy water environment, whereas
in µG, only the D2O was a variable to affect the sperm.
To address this problem, a centrifugal system, such as the NiZeMi
centrifugal microscope could be used to change gravitational vectors
without the need to use H2O or D2O to change
the environment of sperm activation.
Whether the alleviation of restriction to mechanochemical movements
within the sperm flagellum is directly related to the increase in
protein phosphorylation remains to be determined. Such a linkage is
supported by the growing evidence for the involvement of A kinase
anchoring proteins (AKAPs) in the regulation of interactions between
protein kinases, protein phosphatases, and their substrates. For
example, permeable inhibitors of AKAPs inhibited motility of bovine
sperm in a time-dependent manner (27)
, whereas these same
AKAP inhibitors rapidly stimulated motility of sea urchin sperm
(13)
. In a recent collaboration with Stuart Moss
(University of Pennsylvania) we found that sea urchin sperm flagella
contain AKAP82, the same AKAP that comprises the major protein of the
mammalian sperm fibrous sheath (unpublished results). The fact that the
fibrous sheath is not present in sea urchin sperm suggests that
axonemal AKAPs may bind stimulatory second messenger enzymes such as
protein kinase A, whereas the fibrous sheath AKAPs may dock
predominantly inhibitory enzymes such as protein phosphatases. This
idea is supported by recent work in mouse sperm where we identified a
120-kDa protein with the same pT motility-coupled properties as the sea
urchin sperm FP130. However, when the mouse 120-kDa protein increases
in pT content during motility activation, the AKAP82 proteins show a
reduction in pS content (14)
.
Another question raised by the results is whether the changes in sperm
motility and phosphorylation indicate a gravity-sensing mechanism in
these cells. Several studies have demonstrated that sperm tend to swim
in the direction of the gravitational vector (28
,
29
). The mechanism for this may be that gravity acts on
the sperm head as a center of mass and pulls the sperm head toward the
gravitational vector. Once oriented, the tail will continue to propel
the sperm in that direction (28)
. A similar mechanism has
been proposed for the gravitropic response in plants but on a smaller
scale. Gravitational forces acting on the plastid mass interact with
microtubule and actin-based cytoskeletal elements causing directional
modifications on the direction of root growth (30-32)
.
Furthermore, the gravitropic response appears to be mediated through
enzymes that directly regulate protein phosphorylation
(33
, 34
). Data on sperm would suggest that
µG would also produce changes in protein phosphorylation associated
with altered geotaxis in plants.
In conclusion, these results demonstrate that sperm motility activation
in µG is accompanied by significantly faster phosphorylation of
flagellar phosphoproteins, which is consistent with earlier
observations that sperm swim faster in µG. The axoneme-bound
phosphoprotein, FP130, is a primary target of the signal transduction
pathways targeted in µG. In addition, FP130 is a target for speract,
but the temporal pattern of FP130 phosphorylation and dephosphorylation
is altered in µG. Whether these changes impact the rate and
efficiency of egg fertilization in µG is an important question that
warrants further investigation.
|
ACKNOWLEDGMENTS
|
|---|
This study was supported by Grant NAG-2-1016 from NASA (J. S. T.) and Grant HD-33994 from NIH to the Center for Reproductive
Sciences. The outstanding support and coordination efforts of the ESA
Biorack mission Scientists Enno Brinckmann, Ph.D. (STS-81) and Claude
Brioullet, Ph.D. (STS-84), and Biorack Project Manager Peter Genzel,
and NASA/Ames support team members Ron Schaefer, Ph.D., Julianna
Fishman, and Tad Savage are gratefully acknowledged. Special thanks to
CNES/COMAT for the flight hardware, Bionetics Corporation for their
excellent support of the laboratory facilities at KSC, and Eve Stavros
in the POC at JSC for ensuring that we had continuous video downlink
from the orbiter during our flight experiments. We thank Jennifer
Fritch for her excellent technical pre and post-flight support and Mary
Landis and Lorraine Stutzman Tash for their technical assistance during
our pre-flight preparations at KSC. We thank Mission Specialists John
Grunsfeld, M.D (STS-81) and Ed Liu, Ph.D. (STS-84) for their excellent
work performing the experiments aboard space shuttle
Atlantis, and ESA Biorack ground technicians Ulrich Kuebler
(STS-81), Norbert Hiesgen (STS-81 and STS-84), and Didier Narbonne
(STS-84) for their excellent work performing the Biorack ground
experiments at KSC.
|
FOOTNOTES
|
|---|
2 Abbreviations: µG, microgravity; cAMP,
adenosine 3',5'-monophosphate; cGMP, guanosine 3',5'-monophosphate;
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;
KSC, Kennedy Space Center; JSC, Johnson Space Center; STS, Space
Transport System; PTCU, passive thermal conditioning unit; MET, mission
elapsed time (time after launch); ASW, artificial seawater; ESA,
European Space Agency; IOD, integrated optical density; CASA,
computer-assisted sperm analysis; EMA, Experiment Monitoring Area.
Received for publication September 15, 1998.
Revision received October 1, 1998.
|
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TOP
ABSTRACT
INTRODUCTION
P >0.005). At 1 G, the level of FP130
phosphorylation at 60 s were significantly different in the
absence and presence of speract
(§P >0.03).