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Hamsterspermmotility
transformationduringdevelopment
ofhyperactivationinvitroand
epididymalmaturation
ArticleinGameteResearch·January1988
DOI:10.1002/mrd.1120190106
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SusanSSuarez
CornellUniversity
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Gamete Research 1951-65 (1988)
Hamster Sperm Motility Transformation
During Development of Hyperactivation
In Vitro and Epididymal Maturation
Susan S. Suarez
Department of Reproduction, School of Veterinary Medicine, University of California, Davis
The transformation of hamster sperm motility during capacitation in vitro and during
maturation in the caudal epididymis was analyzed and compared using videomicrography .
Sperm recovered from the distal portion of the caudal epididymis, as well as ejaculated
sperm recovered from the uterus exhibited low amplitude, planar flagellar beating. By 3
hr of incubation under capacitating conditions, the caudal epididymal sperm were swimming in helical patterns apparently produced by significantly increased acuteness of
flagellar bending and by torsion seen as abrupt, periodic turning of the head. By 4 hr, most
sperm were hyperactivated, swimming in circles resulting from asymmetrical, planar
flagellar bending that was significantly more acute than the preceding patterns. When
motility parameters of fresh sperm were compared with those of sperm swimming in the
transitional helical pattern and with hyperactivated sperm, transitional sperm had significantly higher net and average path velocities than the others, indicating that they covered
space at the greatest rate. This suggests that the transitional phase plays an important role
in sperm transport. Sperm recovered from the proximal region of the caudal epididymis,
near the corpus, swam in either the helical or hyperactivated patterns, or a mixture of the
two. The means of their flagellar curvature ratios and linear indices were intermediate
between helical and hyperactivated mean values. Thus, sperm undergoing final maturation
in the caudal epididymis reverse the pattern of development of hyperactivation. Also, the
development of hyperactivated motility must therefore entail induction of a preexisting
potential for flagellar movement, rather than a maturational process.
Key words: hamster sperm motility transformation, hyperactivation, in vitro maturation
INTRODUCTION
At some point between insemination and fertilization, many mammalian sperm
are known to undergo a radical transformation in motility that has been termed
hyperactivation [Yanagimachi, 19811. The transformation involves significant quantitative changes in flagellar beating parameters, including increased acuteness of flagellar bends, increased asymmetry of beating and decreased beat and roll frequencies
that give the visual impression of a dramatic qualitative change [Yanagimachi, 1970;
Received April 14, 1987; accepted August 21, 1987.
Address reprint requests to Susan S. Suarez, Department of Reproduction, School of Veterinary
Medicine, University of California, Davis, CA 95616.
0 1988 Alan R. Liss, Inc.
52
Suarez
Katz et al., 1978; Cummins, 1982; Yanagimachi et al., 1983; Cooper, 1984; Suarez
et al., 1983, 1984; Burkman, 19841. Hyperactivation is thought to serve the functions
of enabling sperm to reach the site of fertilization and to penetrate the vestments of
the egg. Its means of serving these hypothesized functions are increased flexibility of
movement and increased capability for generating thrust against solid objects such as
the zona pellucida [Katz et al., 1978; Suarez et al., 1983; Isijima and Mohri, 19851.
Hamster sperm develop hyperactivated motility prior to undergoing the acrosome reaction [Llanos and Meizel, 19831. Their hyperactivated swimming pattern is
circular, resulting from planar asymmetrical flagellar beating. The more acute bend,
called the principal bend, is in the same orientation as the curve of the hooked head.
The less acute reverse bend is in the opposite orientation. After completing the
acrosome reaction, the principal bend is so acute that sperm often complete a circle
in two beats, thereby creating a figure eight pattern [Yanagimachi, 1981; Suarez et
al., 19841. The development of hyperactivation in the hamster occurs over a period
of hours in vitro [Llanos and Meizel, 1983; Katz et al., 19861, but the details of the
sequence and timing of changes in flagellar beating parameters have not been closely
observed. Such information could lead to an understanding of the mechanisms regulating hyperactivation as well as the relationship of hyperactivation to capacitation.
The relationship of the flagellar beating patterns of capacitating sperm to that of
sperm developing motility in the epididymis also has not been studied. Observation
of the motility of sperm recovered from various regions of the epididymis indicates
that many mammalian sperm acquire the ability for circular movement prior to
acquiring the ability to swim progressively [rabbit: Gaddum, 1968; rat: Fray et al.,
1972; bovine: Acott et al., 19831. The circular movement reported to occur in these
species may be identical in pattern with hyperactivation; however, this has not been
investigated. Thus, the present study was also undertaken in order to compare
development of progressive motility during epididymis maturation in the hamster
with the development of hyperactivation during capacitation.
The hamster was chosen as the model system for studying the development of
hyperactivation in vitro and the development of progressive motility in the epididymis
because hamster sperm can be capacitated, hyperactivated, and acrosome-reacted in
a relatively simple defined medium. Furthermore, the acrosomal status of these large
sperm can be ascertained from videotaped images of motile sperm. Finally, golden
hamster sperm develop a relatively simple circular swimming pattern during hyperactivation that is amenable to analysis, whereas sperm of some other species, such as
the mouse, swim in a more erratic pattern when hyperactivated [Cooper, 1984; OldsClarke, 19861.
MATERIALS AND METHODS
Materials
Sexually mature male and female golden hamsters (Mesocn'cetusaurutus) were
purchased from Simonsen Laboratories, Inc. (Gilroy, CA) and maintained under a
14-hr light: 10-hr dark diurnal cycle, with lights-on at 2100. All inorganic chemicals
were purchased from Mallinckrodt, Inc. (Paris, KY) and organics from Sigma
Chemical Co. (St. Louis, MO) , except for N-2-hydroxyethylpiperazine- "-2-ethanesulfonic acid (HEPES buffer) and Fraction V bovine serum albumin (BSA), which
was from Calbiochem, Behring Diagnostics (La Jolla, CA).
Hamster Sperm Motility
53
The medium was modified from that developed for hamster capacitation by
Meizel [Mrsny and Meizel, 19811by adding 25 mM of HEPES buffer and readjusting
the osmolarity by lowering the NaCl concentration. It contained 25 mM HEPES, 110
mM NaC1, 5 mM KCL, 0.36 mM NaH2P04, 24.9 mM NaHC03, 2.4 mM CaC12,
and 0.49 mM MgC12. To these salts were added 6.25 mM L( +)-lactic acid, 0.125
mM Na pyruvate, 5 mM a-D(+)glucose, and 0.5 mM hypotaurine, and 12 mg/ml
Fraction V bovine serum albumin (BSA). Epinephrine was omitted and hypotaurine
was used in place of taurine in order to maximize hyperactivation and minimize the
occurrence of acrosome reactions [Meizel et al., 19801, which would affect motility
parameters [Suarez et al., 19841. The pH was adjusted to 7.65 with 1 N NaOH. It fell
to 7.5 under 5 % C02 in air atmosphere. The medium was filtered through a 0.22 pm
Millex-GV filter, using a Monoject plastic disposable syringe prewashed to remove
toxic barrel grease.
Epididymal Sperm
Male hamsters were killed by COz inhalation and cervical dislocation and
quickly transferred to a 37°C chamber. The caudal epididymides were removed.
Sperm were obtained by puncturing the tubule in the proximal and distal portions of
each epididymis. The thick fluid appearing at each puncture was immediately transferred by fine forceps to 1 ml of 37°C medium in a 1.5-ml polypropylene Eppendorf
centrifuge tube. The motility of the samples from each side was assessed, and the
best samples were used. Sperm from the proximal caudal epididymis (ProxCE sperm)
were videotaped 5-10 min after recovery to allow for dispersal. Their numbers were
1-2 x 106/ml. Sperm from the distal segment (DistCE sperm) were allowed to
disperse for 10 min. Their numbers were determined and adjusted to 2 X 106/mlwith
additional medium and a sample was videotaped. They were incubated in a 1 ml
volume in an uncapped Eppendorf tube in a 5% C02 in air atmosphere at 37°C.
Samples were videotaped after 3 and 4 hr incubation.
Uterine Sperm
Ejaculated sperm were obtained from the uterus using a modification of the
method of Drobnis et al. [1986]. The female was placed in the cage of a male at
0900, which was 2 hr after the onset of darkness and about 2-4 hr prior to the
expected time of ovulation for estrous females. If she indicated the onset of estrus by
showing lordosis and allowing the male to mount, they were left alone for 20 min,
which is about the minimal time required for completion of coitus [Magalhaes, 19701.
Afterwards, the female was retrieved and killed within 10 min. In a 37°C chamber,
the uterine horn was exposed by a midventral incision, grasped with a tweezer at its
cervical end to occlude the lumen and then pierced near the uterotubal junction with
a Pasteur pipet having a tip firedrawn to about half its original diameter. Sperm were
recovered in 0.2 ml medium and transferred to an Eppendorf tube containing 1 ml of
medium. The process was repeated on the opposite uterine horn. The sample containing the highest percentage of motile sperm was then videotaped.
Videotaping and Analysis
Slide chambers were prepared by adhering strips of Parafilm (American Can
Co. , Greenwich, CT) to slides and coating the area between the Parafilm on the slides
and the coverslips with silicone (Sigmacote, Sigma Chemical Co., St. Louis, MO) to
54
Suarez
minimize sticking of sperm to glass. The chamber created by the Parafilm was
approximately 100 pm deep [Suarez et al., 19831. Forty-microliter samples of sperm
suspensions were placed in prewarmed chambers, which were then transferred to a
microscope stage kept at 37°C by an Arenberg Sage Air Curtain (Jamaica Plain,
MA). At least ten microscope fields per slide were videotaped over a period of about
2 min through a 40 X Zeiss phase contrast objective and red filter. A highly lightsensitive microscope video camera (Cohu 5300, Carson Optical Instruments, Oakland, CA) was employed. The image of the sperm and a record of elapsed time in
0.01-sec intervals were recorded simultaneously at a rate of 30 framedsec on a
Panasonic AG-6300 video cassette recorder.
For most samples, the first free-swimming sperm to enter each of the ten
microscope fields was analyzed. In addition, for DistCE sperm, the first sperm per
field that was swimming in a helix was analyzed after 3 hr of incubation and the first
circling sperm per field was analyzed after 4 hr incubation. The helical pattern is
characterized by three-dimensional flagellar beating and is illustrated in Figure 2. A
total of 50 sperm were analyzed per experiment (30 DistCE, 10 ProxCE, 10 uterine)
and 5 replicate experiments were analyzed. The parameters analyzed were flagellar
beat frequency (beatshec) flagellar curvature ratio, average path velocity, and net
velocity (Fig. 1). The flagellar curvature ratio was determined as the straight-line
distance from the headhidpiece junction to the first geometric inflection point of the
tail, divided by the curvilinear distance between these two points [Suarez et al. , 19831.
Net and average path velocities were determined from tracings of the sequential
positions of the headhidpiece junction over 1 sec of time (30 frames). The linear
index [Tessler and Olds-Clarke, 19851 was derived by dividing the net velocity by the
Fig. 1. Measurements. This is a tracing of the videotaped movement of a DistCE sperm that had been
incubated in vitro for 1 hr. The sperm head and flagellum are illustrated as solid lines. The positions of
the headhidpiece junction in successive frames of videotape are shown as points (60 framedsec). The
flagellar curvature ratio is the straight line distance from the headhidpiece junction (b) to the first
geometric inflection point in the flagellum (a), divided by the distance between the two points measured
along the flagellum. The length of the average path traveled in 1 sec is indicated by the curved dashed
line from b to c, while the net distance traveled in 1 sec is the straight dashed line connecting b and c.
The linear index is the net distance divided by the length of the average path.
Hamster Sperm Motility
55
.-..!
Fig. 2. Movement patterns of hamster sperm. The principal bend of the flagellum is indicated by a
solid line and the reverse bend by a dashed line. Successive positions of the headhidpiece junction in
li60-sec intervals are indicated by points connected by straight lines in (a) and (c), where the movement
of the head is confined to a plane. In b, the orientations of the turning head are shown: a: The swimming
pattern of fresh DistCE sperm and sperm recovered from the uterus shortly after coitus. Some sperm
also rolled periodically, resulting in a reorientation of the swimming trajectory. The rate of rolling varied
considerably among the sperm. b: The helical motility pattern of DistCE sperm after 3 hr of incubation
in vitro and of some fresh ProxCE sperm. The points of rapid turning of the head are indicated by
arrows and the position of the flagellum during an abrupt turn is indicated by a dotted line. c: The
circling pattern of hyperactivated DistCE sperm and some fresh ProxCE sperm.
average path velocity. In this study, the average path velocity was determined visually
as the midpoint of the lateral oscillations of the sperm head [Suarez et al., 19831,
rather than as a mathematically derived five-point moving average of a digitized track
[Tessler and Olds-Clarke, 19851.
The data was analyzed by analysis of variance. Individual paired comparisons
were then made as an F test by dividing the mean square of the treatment effect for
the pair by the error mean square from the overall analysis of variance. A significance
level of 0.01 with 1 degree of freedom as the numerator and the overall error degrees
of freedom as the denominator was selected as suggested by Sokal and Rohlf [1981]
for multiple planned comparisons. The data for the uterine sperm were compared
with DistCE sperm in a separate analysis of variance, because the uterine experiments
were run separately and therefore contributed additional sources of variation. The
linear index and flagellar curvature ratio data was normalized for analysis of variance
by the arcsindx transformation for percentage data [Sokal and Rohlf, 19811.
For the tracings used to create Figure 2, a sixth replicate was performed and
videotaped through lox and 25X Leitz phase contrast objectives by a Tritronics
shuttered high-speed video camera (Burbank, CA) in 1/500-secexposures onto a 3/4in videotape at a rate of 60 fieldshec by a JVC U-matic video casette recorder (model
CR06600U, Victor Co. of Japan). When replayed on a field-by-field basis, these
recordings provided greater resolution of the distal portion of the flagellum and
rotation of the head.
56
Suarez
RESULTS
Qualitative Observations
Freshly dispersed DistCE sperm appeared to beat rigidly in real time, because
their flagellar beat was rapid, planar, and of low amplitude (Le., high flagellar
curvature ratio). The low amplitude, planar, slightly asymmetric beat produced
curved trajectories (Fig. 2a). The principal bend originated in the flagellumjust below
the headhidpiece junction and propagated to the end piece. As the principal bend
moved down the flagellum, a less acute reverse bend appeared behind it. At this time
point, rolling occurred in 22% of the analyzed fresh DistCE sperm at the low rate of
approximately one roll per second (less than 1 roll per 13 flagellar beats), which
mainly resulted in reorienting the curved path. A few sperm rolled more frequently.
During the first hour of incubation in vitro many DistCe sperm underwent headto-head agglutination. By about 2 hr they had dispersed. This phenomenon precluded
quantitative analysis of motility during this period. Flagellar bending appeared to
become more acute with time and the incidence of rolling increased, so that between
2 and 3 hr, most sperm were swimming in helical patterns. Although there were
irregularities in the pattern of beating, generally the flagellum produced two principal
bends with the head oriented in one direction, then the head turned about 180" prior
to the initiation of the next bend (Fig. 2b). Turning was not gradual, but seemed to
occur rather abruptly during a particular phase of the flagellar beat cycle.
By 4 hr, most sperm were hyperactivated, swimming in small circles resulting
from acute, asymmetrical planar flagellar bending ,(Fig. 2c). The hyperactivated
motility pattern was identical with that observed in hamster sperm prior to undergoing
the acrosome reaction in vitro [Suarez et al., 19841. In fact, the percentage of motile,
acrosome-reacted sperm after 4.5 hr of incubation in the last two experiments were
69% and 90%. Few acrosome-reacted sperm were observed at 4 hr. They were
excluded from analysis, since they have been shown to bend in significantly more
acute angles than unreacted sperm [Suarez et al., 19841.
Sperm retrieved from the uterus within 10 min of coitus swam in a pattern that
appeared identical to that of fresh DistCe sperm (Fig. 2a). They, too, showed planar,
low-amplitude beating that was even less frequently interrupted by rolling (only 8%
of the analyzed sperm rolled). When incubated in vitro under the same conditions as
epididymal sperm, they underwent a similar sequence of motility pattern transformation and became hyperactivated.
Sperm recovered from the proximal caudal epididymis, close to the corpus of
the epididymis, swam in either the helical pattern of DistCE sperm that had been
incubating for 3 hr, the hyperactivated pattern of sperm that had been incubating for
4 hr, or a mixture of the two patterns. Sperm recovered from the corpus or adjacent
caput epididymis flexed their flagella in acute bends that were often in the opposite
direction with regard to the orientation of the head than the acute bends formed by
hyperactivated sperm. Unfortunately, these sperm were rather sluggish and did not
survive incubation. For this reason, their motility was not analyzed quantitatively.
When ProxCE sperm were incubated under identical conditions as DistCE sperm, the
pattern of their motility was retained, and the percent of motile sperm did not fall
noticeably in 4 hr, but they moved more slowly with increasing incubation time. They
also began to agglutinate in head-to-head fashion shortly after recovery and later
dispersed.
Hamster Sperm Motility
57
The percentages of sperm from the uterus, DistCE, and ProxCE that were
swimming in each pattern is summarized in Figure 3, referring to the patterns
illustrated in Figure 2. The patterns of fresh DistCE sperm are either “planar” or
“rolling,” the latter consisting of the planar pattern with rolling superimposed upon
it. This pattern is not illustrated because the rolling occurred at various rates and
actually created a multitude of patterns. The percentages of sperm in each of the four
categories of swimming patterns were determined by categorizing the swimming
behavior of the sperm that were on the initial video frame of each microscope field
taped during the 2-min period.
Motility Analysis
Although it was difficult to assess accurately motility of DistCE sperm during
the first 2 hr because of agglutination, subjective assessment indicated that the proportion of motile sperm averaged 80% throughout the entire incubation period.
In order to examine the measurable changes in flagellar motion that were
responsible for the qualitative changes observed during the development of hyperactivated motility in vitro, the motility parameters of samples of fresh DistCE sperm
were compared with those sampled from the same tubes after 3 and 4 hr of incubation.
At 3 hr, helical motility predominated, so data was obtained from sperm swimming
in these patterns. At 4 hr, hyperactivated (circular motility) sperm was measured.
The flagellar curvature ratios and linear indices were significantly reduced in the
helical sperm when compared with the fresh sperm and also in the hyperactivated
sperm when compared with the helical sperm (Figs. 4,5;Table 1). The flagellar beat
frequency decreased with the development of hyperactivation (Fig. 6; Table 1) It was
not possible to compare sperm swimming in different patterns within the same time
Fresh, Planar
Fresh, Rolling
277
213
Helical
Hmractivated
243
T
168
T
310
T
Uterine
DistCE:Ohr
DistCE:3hr DistCE:4hr
ProxCE
Fig. 3 . The percentage (+ SEM) of uterine, DistCE, and ProxCE sperm swimming in each of four
patterns. The patterns are described in Results and illustrated in Figure 2. The percentages were
determined by categorizing all of the sperm that appeared in the first videoframe of each microscope
field taped during a 2-min period. The numbers above each bar represent the numbers of sperm
categorized.
58
Suarez
S
2 80c
E
-
g, 70
3
c
$
J
u
--z
-5
.c
60-
-
-I
50
-
40
30
I
uterine fresh
I
helical
I
hyper ProxCE
DistCE
Fig. 4. The flagellar curvature ratios ( X 100) of ejaculated and caudal epididymal hamster sperm. Each
symbol, with standard error bars, represents the mean of 10 sperm analyzed for a particular male.
Uterine: Ejaculated sperm collected from the uterus shortly after coitus. Fresh: DistCE sperm less than
15 min after collection. Helical: DistCE sperm incubated for 3 hr in vitro that swam in a helical pattern.
Hyper: DistCE sperm incubated for 4 hr that were circling. ProxCE: ProxCE sperm 5 min after
collection.
i
X
X
E
.- m /
20
I
uterine fresh
f
I
I
I
helical
DlstCE
hyper 3roxCE
Fig. 5. The linear indices ( X 100)of ejaculated and caudal epididymal hamster sperm (means
Please refer to legend for Figure 4.
SEM).
Hamster Sperm Motility
59
TABLE 1. A Summary of the Results of the Motility Analysis*
DistCE
Flagellar curvature
ratio ( x 100)
Linear index ( X 100)
Beat frequency
(beatdsec)
Net velocity
(pm/sec)
Average path velocity
(pmisec)
Helical
Uterine
Fresh
94 f 0.6a
91 f 1.9b
+
3.6a
14 k 1.6a
83
82 f 2.8a
14 i- 1.8a
123 5 7.2a
Hyperactivated
+ 3.6"
60 + 2.7b
ProxCE
56
44 f 1.4d
52
12 & 1.7b
29 f 2.3'
8 + 2.3'
50 f 5.0d
7 f 1.2d
156 & 4.9b
63 f 5.7'
152 5 12.2a 266 & 12.9b
j~ 3.6'
223 f 10.6'
*Each number represents the mean (fSEM) for 50 sperm. Within each line, different superscripts
indicate that the means were significantly different (p<0.01). Uterine sperm were compared with fresh
DistCE sperm in a separate analysis because they were collected at different dates from the epididymal
sperm.
16
14t
12 10
I
-
8 6-
4
I
d
uterine fresh
I
helical hyper ProxCE
DistCE
Fig. 6. The beat frequencies, in flagellar beatdsec, of ejaculated and caudal epididymal hamster sperm
(means _+ SEM). Please refer to legend for Figure 4.
point, except to compare helical and hyperactivated DistCE sperm at 4 hr of incubation. In this comparison, helical sperm still had significantly greater beat frequencies,
linear indices, and net and average path velocities (Table 2). Only the flagellar
curvature ratios were not significantly different.
In contrast to the trend shown in linear index, a derivative parameter, both net
and average path velocities peaked in the transitional sperm that swam in the helical
pattern (Figs. 7,8; Table 1). Since both of these velocity parameters were significantly
higher among transitional (helical pattern) sperm when compared with fresh sperm
and hyperactivated sperm, the transitional sperm therefore cover space at the greatest
rate.
ProxCE sperm had flagellar curvature ratios and linear indices that were significantly lower than that of DistCE sperm measured shortly after recovery. As expected
by the observed mixture of helical and circular patterns apparent in the ProxCE
Suarez
60
TABLE 2. A Comparison of the Motility Parameters of Helical and Hyperactivated DistCE Sperm
Incubated for 4 h r In Vitrot
DistCE at 4 hr
Helical
Flagellar curvature ratio ( X 100)
Linear index ( X 100)
Beat frequency (beatdsec)
Net velocity (pmisec)
Average path velocity (Kmhec)
54
52
12
157
259
Hyperactivated
f 3.2*
47
35
9
65
219
i 7.5**
& 0.4**
k 9.3**
f 13.8**
k 1.4*
f 2.6**
& 0.3**
k 6.4**
f 8.6**
?In this case, the first sperm entering each of 10 fields was analyzed, then categorized as helical or
hyperactivated. Any sperm that turned about its long axis (Le., rolled) was considered to be helical. Out
of 50 sperm analyzed, 16 were categorized as helical. Means SEM are reported.
*p<O.l(not significant).
**p<O.Ol.
200
150
-
100
-
50
-
Q)
v)
2
2
I
H
I
t
Q)
>
CI
0
I
I
I
I
Fig. 7. The net velocities, in pm/sec, of ejaculated and caudal epididymal hamster sperm (means k
SEM). Please refer to legend for Figure 4.
samples, the means were intermediate between those of DistCE sperm swimming in
transitional (helical) and hyperactivated (circular) patterns. The beat frequencies of
the ProxCE sperm were significantly lower than all varieties of DistCE sperm.
Nevertheless, they did vary widely, probably reflecting a sensitivity to manipulation
in vitro, rather than a physiological state.
The beat frequencies and linear indices of uterine sperm were not different from
those of fresh DistCE sperm; however, their flagellar curvature ratios were higher,
indicating less acute bending.
In the three experiments in which rolling fresh DistCE sperm were analyzed,
no significant differences were detected in flagellar beating parameters between
rolling and nonrolling sperm: the mean (+SEM) flagellar curvature ratio for rolling
sperm was 0.90+0.018 and for nonrolling sperm was 0.91 f0.013, while the mean
beat frequency for rolling sperm was 13.8k0.25 and for nonrolling sperm was
13.6 0.84.
+
Hamster Sperm Motility
100
'
I
fresh
I
I
helical
hyper
61
Fig. 8. The average path velocities, in pm/sec, of ejaculated and caudal epididymal hamster sperm
(means SEM). Please refer to legend for Figure 4.
+
DISCUSSION
Initially, DistCE sperm exhibited planar, low-amplitude flagellar beating. Since
this movement pattern differs from that observed for ejaculated sperm of many other
species by the absence of rolling [Cumins, 1982; Suarez et al., 1983; Hafez, 19871,
ejaculated sperm were collected from the uterus shortly after coitus for comparison.
The pattern of motility was identical in appearance and neither DistCE nor ejaculated
sperm rolled. In addition, the ejaculated sperm collected from the uterus underwent
apparently identical development of hyperactivated motility when incubated in vitro.
Thus, the swimming pattern of fresh DistCE sperm is that of ejaculated sperm.
Yanagimachi [ 19701 and Ishijima and Mohri [ 19851 also described a nonrolling
movement pattern in fresh hamster caudal epididymal sperm. In this study, the effect
of confinement of the 170-pm-long hamster sperm in 100-pm-deep chambers was
considered. Similarly confined rabbit ejaculated sperm are known to be inhibited
from rolling [Suarez et al., 19831. The fresh DistCE hamster sperm were placed in 1
mm-deep, silicone-coated chambers. Most still traced large, flat circles, and rolling
was still rare throughout the sample; therefore, confinement did not seem to be the
cause of planar flagellar movement.
Rolling began to develop among the DistCE sperm during incubation in vitro
and was periodic in the 3-hr transitional sperm. Rolling has been attributed to
rotational change in the plane of flagellar bending that occurs during bend propagation
[Woolley and Osborn, 1984; Yeung and Woolley, 19841; however, nothing is known
of the mechanisms producing this phenomenon. The observation that rolling is
initiated then terminated during in vitro capacitation in the hamster raises some
interesting questions. For example, is rolling a separately controlled phenomenon
from flagellar bending acuteness and asymmetry? Is it initiated or suppressed under
different conditions in different species because of difference in the composition of
cell surface receptors, ion channels or masking factors? In the mouse, hyperactivated
motility is less progressive than fresh DistCE sperm motility and is characterized by
increased acuteness of flagellar bending, but the path of the sperm is not completely
62
Suarez
circular [Cooper, 1984; Neil1 and Olds-Clarke, 1986; Suarez and Osman, 19871.
When videotapes of mouse sperm hyperactivated by incubation in vitro [Suarez and
Osman, 19871 or by exposure to calcium ionophere A23187 [Suarez et al., 19871
were reviewed, it appeared that the lack of continual circular movement is attributable
to rolling. In the future, then, the investigation of hyperactivation may benefit from
dissecting the study of sperm movement into the components of rolling and acuteness/
asymmetry.
Another curious aspect of rolling of the head is that it is not a continuous
process. When examined closely, the helical pattern observed was apparently generated by two flagellar beats initiated within a plane, followed by an abrupt 180” turn
of the head. While there were variations in the frequency of head turning, this pattern
predominated in the samples incubated for 3 hr. It has been reported that the rate of
rotation in hamster sperm is independent of the rate of bend initiation, and that threedimensional beat cycles may be interrupted by planar beat cycles [Woolley and
Osborn, 1984; Yeung and Woolley, 19841. In the present experiments, the relationship
between the occurrence of turning and other flagellar beat parameters were investigated. While the flagellar curvature ratios and beat frequencies differed significantly
between transitional and hyperactivated sperm, no significant difference was detected
between fresh DistCE sperm that rolled and those that did not. These results indicate
that rolling is controlled by a separate mechanism from that which controls the
acuteness of flagellar bending and the rate of bend initiation. This is quite possible,
because work with demembranated sea urchin sperm indicates that various flagellar
bending parameters are controlled by separate mechanisms [Brokaw, 19831.
The results reported herein differ from those obtained by Katz and coworkers
119861, who reported an “invigoration” (referring to an increase in beat frequency)
occurring during capacitation in vitro of DistCE hamster sperm. The reason for the
difference might derive from the fact that their fresh sperm had been washed once
and evaluated in a phosphate-buffered saline/sucrose solution. Subsequently, they
were passed through a column of glass beads to remove dead sperm, then incubated
in capacitation medium. Invigoration could be attributable to recovery from this
pretreatment. The present study was designed to minimize trauma to the sperm. The
sperm were simply diluted in capacitation medium, rather than being washed, and
were never allowed to cool. Also, HEPES buffer was added to the medium to dampen
pH elevation that occurs when sperm are exposed to air lacking 5% C02. The
presence of small amounts of epididymal fluid in this incubation medium apparently
did not block hyperactivation or even capacitation, since acrosome reactions were
observed to occur within the same time frame as those of washed hamster sperm
[Llanos and Meizel, 19831. Thus invigoration of beat frequency is not an integral part
of the hyperactivation process.
The most surprising result of this study was the fact that helical DistCE sperm
had greater net and average path velocities than both fresh, nearly linear sperm and
circling, hyperactivated sperm. To verify that the drop in velocities between helical
and hyperactivated sperm was not likely to be attributable to aging, the beat frequencies and velocities of helical and hyperactivated sperm incubated for 4 hr were
compared (Table 2). Even at 4 hr, these measurements were significantly higher in
helical sperm than in hyperactivated sperm. When considered alone, net velocity does
not provide an accurate assessment to the movement of sperm (e.g., both a dead
sperm and a circling sperm could have a net velocity of zero), but when combined
Hamster Sperm Motility
63
with average path velocity in this case, it indicates that helical sperm have the greatest
potential for covering space. Because of the constraint of measuring in only two
dimensions, the average path velocity data for helical paths are underestimates of the
true average path velocities; therefore, these sperm are covering space at an even
greater rate than indicated by the data. Since transitional sperm cover space at the
greatest rate, this phase could play an important role in sperm transport. Although it
might be argued that the lateral component of movement would not transport the
sperm any closer to the egg, the coiling of the hamster oviduct and the folding of
oviductal mucosa do not present the sperm with a linear route. While it remains to be
tested experimentally, later movements may aid navigation under these conditions.
Mouse sperm trapped in mucosal pockets were observed to escape after producing
deep flagellar bends that changed the orientation of the head [Suarez, 19871.
When the motility of ProxCE sperm was compared to that of DistCE sperm
incubated to hyperactivate in vitro, it was apparent that the ProxCE sperm swam in a
pattern identical with that of either the hyperactivated DistCE sperm or the sperm in
the transitional state at 3 hr, or they swam in a mixture of the two patterns. This was
reflected in the mean linear indices and flagellar curvature ratios, which were
intermediate in values between those of helical and hyperactivated DistCE sperm.
The beat frequencies of ProxCE sperm were lower, however, than those of DistCE
sperm and continued to decrease with time despite the maintenance of the swimming
pattern. The sluggishness of ProxCE sperm might be attributable to their need for
additional substances in order to remain motile. It has been shown that hamster sperm
from the caput epididymis become and remain motile in the presence of a phosphodiesterase inhibitor and seminal plasma [Cornwall et al., 19871. Demembranated
hamster caput epididymal sperm can be activated by CAMP and ATP, even though
intact sperm are only weakly motile [Ishijima and Mohri, 19851. In spite of the lesser
vigor of ProxCE sperm, it was apparent that they undergo the reverse of the process
of hyperactivation undergone by mature, capacitating sperm. Thus, the development
of the hyperactivated flagellar bending pattern in the female tract is not a maturational
process but rather an induction of a preexisting potential.
CONCLUSIONS
In summary, hamster sperm motility in the caudal epididymis develops during
maturation in reverse order to the pattern of development of hyperactivated motility
during capacitation in vitro. During the development of hyperactivated motility in
vitro, sperm in the transitional phase show greater potential for covering space than
freshly ejaculated or hyperactivated sperm. The helical movement pattern of the
transitional phase results from moderately acute flagellar bending punctuated periodically by abrupt turning of the head.
ACKNOWLEDGMENTS
This work was supported by NIH research grant HD19584. The author thanks
Wayne I. Gottlieb and Richard A. Osman for helpful discussion and review of the
manuscript and Dr. Irwin K. Liu for the use of his high-speed video system.
64
Suarez
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