Andrologia 30: 153-157, 1998
WAVE
PARAMETERS OF THE SPERM FLAGELLUM AS PREDICTORS OF HUMAN SPERMATOZOA MOTILITY
O. Vera1, M.G. Muñoz2 and
K. Jaffe2
1Facultad de Ciencias
Veterinarias, Universidad Central de Venezuela, Maracay, Venezuela.
2 Departamento de Biología de
Organismos, Universidad Simón Bolívar, Apartado 89000, Caracas 1080, Venezuela
Short title: Wave
movement of the sperm flagellum
Key Words: sperm
movement, motility, spermatozoa, wave
Klaus Jaffe
Departamento
de Biología de Organismos, Universidad
Simón Bolívar. Apartado 89000,
SUMMARY
We characterized the undulatory movement of the sperm’s flagellum as a sigmoid
wave by measuring the absolute and linear speed of the sperm, period, amplitude
and length of the flagellum’s wave, and the segment comprised between the head
and the origin of the movement in the flagellum. These parameters were
correlated with traditional ones used to determine the pattern of movement of
the sperm. Our results show that wave parameters are useful predictors of sperm
motility. They correlate among themselves, and thus, a few wave parameter may characterize the sperm motility. The advantage
of wave parameters is that they can be easily obtained and can be eventually
associated to the sperms internal morphology.
INTRODUCTION
Sperm motility is thought to be an
important factor in the biology of reproduction at both the systemic and
cellular levels, and the quality of the sperm movement has been related to
fertility in humans (Dredsner and Katz,1981) and other animals (Suarez et al.,1984). The internal
structure of the flagellum is now well understood but not its consequences on
the flagellum’s undulations, which should be relevant in determining the
quality of the movements (Fauci and Mc. Donald,1995). During the last years, seminal evaluation has been
undertaken using the computerized image analysis of the sperm’s movement. The
computerized system considers the track of the head to classify the different
types of movement of the spermatozoa . The lateral
displacement of the sperm head is a characteristic that seems to be associated
with its capacity to penetrate cervical mucus and progress into the female
genital tract (Aitken, 1995). This parameter is
related to minor changes in
the amplitude of the lateral displacement of the sperm’s head,
which in turn is reflective of the undulatory
movement of the flagellum, all of which determines the propulsive force
necessary for fertilizing the egg (Aitken, 1995).
Considering that the sperm movement
is originated in the flagellum, and thus, that direct measurements of the
parameters of the flagellum’s movement would be more convenient, we attempt
here to characterize directly the undulatory movement
of the sperm’s flagellum as a sigmoid wave by measuring the absolute and linear
speed of the sperm, period, amplitude and length of the flagellum’s wave, and
the segment comprised between the head and the origin of the movement in the
flagellum. These parameters were correlated with traditional ones used to
determine the pattern of movement of the sperm.
MATERIALS AND METHODS
Twelve semen samples were obtained
by masturbation following 3 to 5 days of sexual abstinence, from 12 healthy
donors. For each sample, motile sperms were isolated by overlaying the semen
samples with an equal volume of warm BWW (Brinster
1965), supplemented with 0.3% Bovine Serum Albumin, incubating the mixture at
37° C for 60 min and then collecting the BWW medium sperm suspension. Each
sample was analyzed with two different methods:
1- A semi
automatic system (Comhaire & Vermeulen
1995) consisting in a single step for the assessment of sperm characteristics.
A drawing tube was introduced between the objective and the ocular of the
microscope, to observe motile spermatozoa and to track their movement manually
with the help of a cursor on a digitizing tablet. This tablet generated data
which were analyzed with a computer program (Hintnig
et al. 1988). For each sample, a minimum
of 50 spermatozoa were analyzed. The method allowed for the determination of
sperm concentration, percentage of motility, sperm linear velocity, linear
index, and motility grading. Motility grading had four categories: MG-A: sperms
with rapid linear progressive motility with linear velocity in excess of 22 µm/s; MG-B:
sperms with slow or sluggish linear or non linear movement and with linear
velocity below 22 µm/s, MG-C: sperms with non-progressive motility and velocity
below 5 µm/s: MG-D: immobile sperms (Comhaire & Vermeulen 1995).
2- The sperm suspensions were observed under phase contrast microscope and the
sperm movement were registered with a video camera which was connected to the
microscope. The images were registered with a VHS system. With this method we
analyzed a minimum of 20 sperms per sample (total of 277 sperms) from the 12
different sample preparations. Then the sperm movement was analyzed by assuming
that the flagellum’s movement can be described by a sigmoid wave. The following
characteristics of the wave were measured: amplitude, wavelength, speed and
period. We also measured the length of the segment comprised between the head
and the site of the flagellum where the undulatory
movement was initiated. Only sperms that exhibited complete undulatory
movement of the flagellum visible on the two dimensional plane of the screen
were analyzed. When a sperm with these characteristics was identified, the VHS
was stopped and the image was used to measure the variables in mm. Figure 1
illustrates the measurements taken. A graduated Neubauer
chamber was used to measure and calculate the correspondence between the values
in mm obtained in the monitor with the real values. The period was defined as
the time necessary to realize a whole flagellum’s wave or one cycle. To estimate the period we fixed an
initial position of the sperm and measured the time taken to describe a whole undulatory movement of the flagellum, i.e. until the wave
returned to the initial position. The period was expressed in seconds / cycle.
The statistical correlation
between the different variables were obtained applying the Spearman rank
order correlation analysis.
RESULTS
The results obtained for the
different variables characterizing the wave movement of the sperm’s flagellum
(Figure 2-6), show
that the variables have discontinuous values. That is, the variables seem to
converge onto discrete values, where intermediate values have a very low
probability to occur. This effect is strongest in variables measuring simple
wave features. The movement of the flagellum rarely starts at its insertion
into the head of the sperm. The site of the flagellum where
the movement starts (i) seems to be related to the
amplitude (a) and length (l) of the wave described by the flagellum.
Consequently the wave parameters and the speed (s) of the sperm is also related to that site. Our results show that all wave
parameters measured correlate among themselves (Table 1). Linear regressions
between the variables (Table 2) may give us an idea how they relate. We found
that the speed of the sperm can be predicted from any of the parameters with a
reasonable precision (correlation coefficient r = 0.895 using i) and that it is inversely related to the period (p).
Using all four variables to predict the speed improves only slightly the
precision (r = 0.908). On the other hand, i can be
predicted from a and l
(r = 0.966) and p from a and l (r = 0.934). That is, the sperms
movement can be described (using length measurements of a, l and i in µm and of p in ms), with the following formula :
i = 0.27 a + 0.14 l (ß
for a = 0.32, l = 0.66)
p = 32 a + 7 l (ß
for a = 0.5, l = 0.45)
s = 6.8 i (ß for i = 0.9)
s = 3 a + 1.2 l - 29
p (ß
for a = 0.47, l = 0.73, p = -0.3)
The
wave parameters measured correlated strongly with the motility indices
calculated with the semi automated analysis system (Table 3), suggesting in
fact that the wave parameters are reliable indicators for sperm motility.
DISCUSSION
Our results suggest that the
flagellum is constricted in the type of movements it can perform to a finite
number of states, probably reflecting the degrees of freedom the morphological
structures allow regarding the expression of movement of the flagellum. This
result seems to be related to the discrete nature of the activation process of
microtubules (Kamimura and Mandelkow
1992), suggesting that the microtubules responsible for the movement of the
flagella have a finite number of combinations in which they can express their
activity. The movement of the flagellum of the sperm can be viewed as a string
vibrating in a viscous medium, or as a flexible screw exerting force on a
viscous medium. These kind of problems are best solved
empirically, as no physical analytical solution to them is known.
David et al. (1981) studied the
amplitude of the lateral movement of the sperm head as a reflective of the
amplitude of the flagellum’s wave. Here we report directly the characteristic
of the flagellum’s movement and correlate them with the physical parameters defining
the sigmoid wave formed by the moving spermatozoa. Our results show that the
wave parameters of the flagellum are useful predictors of sperm motility, as
they correlate significantly among themselves, and thus, a single or a few wave
parameter may characterize the movement of the sperm.
An advantage of wave parameters over
other motility indices is that wave parameters can eventually be associated to
the sperms internal morphology in more direct ways, as they represent real
physically characteristics of a movement. If this can be confirmed
independently, it suggests that wave parameters may be more helpful in relating
sperm movement to sperm pathology and to fertility. One indication of the close
relation between the wave parameters measured and their internal morphology and
physiology is the quantum nature of these values. The wave parameters showed a
wide variability and a tendency to cluster around discontinuous values, where
intermediate values have a low probability to occur. It should be possible to
explain this tendency taking into account the number of microtubules and their
fixed pattern of relative movements (Aitken, 1995),
which would determine the movement pattern of the flagellum. We suggest, thus,
that the discrete distribution of the wave parameters reflects a discrete
number of possible movements of the sperm, restricted by the limited array of
possible interactions among the fixed number of microtubules in the flagellum..
We suggest that the new methodology
described here is a useful tool for the characterization of the motility of
spermatozoa. This method has several advantages :
Wave parameters can be assessed without the help of computers and are thus easier to obtain than
alternative indices ; results obtained in this way are comparable with the
velocity obtained by the semi-automated seminal evaluation system; wave
parameters can be obtain from any static photo of moving sperms and their
measurement is very simple. In addition, automatization of these
measurements is also possible and desirable as it increases the speed and
precision of the measurements. The next step should to be to investigate the
relation between the characteristics of the flagellum’s wave parameters and
fertility.
REFERENCES
Aitken, J. (1995). Cell Biology of Human Spermatozoa. In: Gametes. Chapter 11,
pp: 206-232. J.G. Grudzinskas and
J.L. Yovich (eds.)
Comhaire, F., and Vermeulen, L. (1995). Human Semen Analysis. Human
Reproduction Update. 1(4): 343-362.
Brinster, R.L. (1965). Studies on the development of mouse embryos in vitro. IV. Interaction of energy sources. J. Reprod. Fert., 10: 227-240.
David, G., Serres, C. and Jouannet,
P. (1981). Kinematics of
Human Spermatozoa. Gamete Res. 4:83-95.
Dredsner, R.D. and Katz, D. F. (1981).
Relationships of mammalian sperm motility and morphology to hydrodynamic
aspects of cell function. Biol. Reprod.
25:920-930.
Fauci, L.J. and Mc. Donald, A. (1995). Sperm motility in the presence of boundaries. Bull Math.
Biol. 57: (5):679-699.
Hinting,
A., Schoonjans, F. and Comhaire,
F. (1988). Validation of a single step procedure for the
objective assessment of sperm motility characteristics. Int. J. Androl. 11:227-287.
Kamimura, S. and Mandelkow, E. (1992). Tubulin protofilaments and kinesin-dependent
motility. J. Cell Biol. 118:865-875.
Suarez,
S.S., Katz, D.F. and Meizel, S. (1984). Changes in
motility wich accompany the acrosome
reaction in hyperactived hamster spermatozoa. Gamete
Res. 10:253-265.
Table 1: Correlation coefficients between wave
parameters
+----------+------------------------------------------------------+
| STAT. | Correlations: |
| BASIC | Marked correlations are significant at
p < .05 |
| STATS |
N=277 (Casewise deletion of missing data) |
+----------+----------+----------+----------+----------+----------+
| Variable | Initial |Amplitude
|Wavelength| Period | Speed |
| | segment | | | | |
+----------+----------+----------+----------+----------+----------+
|Initial |
1.0000 | .2364*| .2983*|
-.1443*| .1592*|
|segment | p=
--- | p=.000*| p=.000*|
p=.016*| p=.008*|
+----------+----------+----------+----------+----------+----------+
|Amplitude
| .2364*| 1.0000 |
-.1795*| .2583*| .0115 |
| |
p=.000*| p= --- | p=.003*|
p=.000*| p=.849 |
+----------+----------+----------+----------+----------+----------+
|Wavelength| .2983*|
-.1795*| 1.0000 | -.1494*|
-.0965 |
| |
p=.000*| p=.003*| p= --- |
p=.013*| p=.109 |
+----------+----------+----------+----------+----------+----------+
|Period |
-.1443*| .2583*| -.1494*|
1.0000 | -.4520*|
| |
p=.016*| p=.000*| p=.013*|
p= --- | p=.000*|
+----------+----------+----------+----------+----------+----------+
|Speed |
.1592*| .0115 | -.0965 |
-.4520*| 1.0000 |
| |
p=.008*| p=.849 | p=.109 |
p=.000*| p= --- |
+----------+----------+----------+----------+----------+----------+
Table II: Multiple Regression for the prediction of the speed of movement of
sperm
+----------+-----------------------------------------------------------------+
| STAT.
|Regression Summary for Dependent Variable: SPEED |
| MULTIPLE |R= .90836130 R2= .82512025 Adjusted R2=
.82255791 |
| REGRESS. |F(4,273)=322.02
p<0.0000 Std. Error of estimate: 13.321 |
+----------+----------+----------+----------+----------+----------+----------+
|
| BETA |
| N=277
| | of BETA | |
of B | | |
+----------+----------+----------+----------+----------+----------+----------+
|Init. segm|
.403307 | .099064 | 3.0699 |
.754052 | 4.07116 | .000061 |
|Wavelength| .446764 | .097343 |
.7257 | .158118 | 4.58960 |
.000007 |
|Amplitude | .324978 | .078928 |
2.0465 | .497047 | 4.11741 |
.000051 |
|Period |
-.265624 | .071380
| -26.1439 | 7.025526 | -3.72128 |
.000241 |
+----------+----------+----------+----------+----------+----------+----------+
+----------+-----------------------------------------------------------------+
| STAT. |Regression Summary for Dependent Variable:
SPEED |
| MULTIPLE |R=
.90249818 R2= .81450296 Adjusted R2= .81247197 |
| REGRESS. |F(3,274)=401.04 p<0.0000 Std. Error of estimate:
13.694 |
+----------+----------+----------+----------+----------+----------+----------+
|Amplitude | .471328 | .072236 |
2.9682 | .454903 | 6.52487 |
.000000 |
|Wavelength| .728138 | .070469 |
1.1827 | .114466 | 10.33270
| .000000 |
|Period | -.296823 | .072956 | -29.2147 | 7.180688 |
-4.06851 | .000062 |
+----------+----------+----------+----------+----------+----------+----------+
+----------+-----------------------------------------------------------------+
| STAT.
|Regression for Dependent Variable: SPEED |
| REGRESS. |and each of the wave parameters one by
one |
+----------+----------+----------+----------+----------+----------+----------+
|Init. segm|
.895371 | .026805 |
0.895 | .204033 | 33.40300 | 0.00 |
|Amplitude | .861216 | .030592 | 0.861 |
.192655 | 28.15130 | 0.00 |
|Wavelegth | .886093 | .027900 | 0.886 |
.045319 | 31.75982 | 0.00 |
|Period | .797145 | .036344 | 0.797 | 3.577124 | 21.93350 | 0.00 |
+----------+----------+----------+----------+----------+----------+----------+
Table III: Correlation between wave parameters and sperm
motility indices
as given by Comhaire & Vermeulen (1995)
+----------+------------------------------------------------------+
| STAT. | Correlations |
| BASIC | Marked correlations are significant at
p < .05000 |
| STATS |
N=277 (Casewise deletion of missing data) |
+----------+----------+----------+----------+----------+----------+
| | Initial |Amplitude |Wavelegth
| Period | Speed |
| Variable | segment | | | | |
+----------+----------+----------+----------+----------+----------+
|Sperm |
.3405*| -.0240 | .3458*|
-.2357*| .2669*|
|Concentra.|
p=.000*| p=.691 | p=.000*|
p=.000*| p=.000*|
+----------+----------+----------+----------+----------+----------+
| MG-A | .2960*|
.3101*| -.1788*| -.0729 |
.2356*|
| |
p=.000*| p=.000*| p=.003*|
p=.227 | p=.000*|
+----------+----------+----------+----------+----------+----------+
| MG-B | .0959 |
-.1047 | .0362 | -.1312*|
.3074*|
| |
p=.111 | p=.082 | p=.549 |
p=.029*| p=.000*|
+----------+----------+----------+----------+----------+----------+
| MG-C | -.1618*|
-.0806 | .1904*| .1101 |
-.3824*|
| |
p=.007*| p=.181 | p=.001*|
p=.067 | p=.000*|
+----------+----------+----------+----------+----------+----------+
| MG-D | -.2275*|
.0166 | -.2855*| .1353*|
-.1160 |
| |
p=.000*| p=.783 | p=.000*|
p=.024*| p=.054 |
+----------+----------+----------+----------+----------+----------+
| Sperm |
.2642*| .3661*| -.3310*|
-.0158 | .3329*|
| Speed |
p=.000*| p=.000*| p=.000*|
p=.794 | p=.000*|
+----------+----------+----------+----------+----------+----------+
| Linear |
.2834*| .3452*| -.2988*|
-.0441 | .3497*|
| Speed |
p=.000*| p=.000*| p=.000*|
p=.465 | p=.000*|
+----------+----------+----------+----------+----------+----------+
| Linear |
.1714*| -.1488*| .3144*|
-.1563*| .0599 |
| Index |
p=.004*| p=.013*| p=.000*|
p=.009*| p=.321 |
+----------+----------+----------+----------+----------+----------+
Figure
1 :
Wave parameters of the movement of the flagellum, indicating the amplitude
(a), distance from head where the wave
movement starts (i) and the wavelength (l).
Figure
2 :
Frequency distribution of values for spermatozoa for the distance from the head
where the movement of the flagellum starts
Figure
3 :
Frequency distribution of values for spermatozoa for the wavelength of the
movement of the flagellum.
Figure
4 :
Frequency distribution of values for spermatozoa for the amplitude of the wave
length of the flagellum
Figure
5 :
Frequency distribution of values for spermatozoa for the period of the wave
movement of the flagellum.
Figure
6 :
Frequency distribution of spermatozoa velocities.
