- Email: [email protected]
The inhibition by amytal of respiration and motility of bull spermatozoa
312
Experimental
THE
Cell Research
37, 312-326
(1965)
INHIBITION BY AMYTAL OF RESPIRATION AND MOTILITY OF BULL SPERMATOZOA R.
The Population
Council,
RIKMENSPOEL
The Rockefeller Received
March
Institute,
New
York,
N.Y.,
U.S.A.
16, 1964
recent papers [ 13, 14, 161 apparatus has been described which makes it possible to simultaneously measure, on the same preparation, motility and respiration of sperm suspensions. This system has been developed to investigate relations between the oxidative metabolic pathway as an energy producer for sperm and their motility as an energy consumer. These relations cannot a priori be assumed to exist. Calorimetric measurements by Rothschild [19] of the heat production by anaerobic metabolism of bull sperm gave values of 6 to 9 X lo-6 erg/sperm/set. This is more than one order of magnitude more than estimates by Carlson [3] of the energy which is dissipated by the moving flagella in the surrounding fluid. From data of Rothschild and Cleland [21] on the oxygen consumption of sea urchin sperm, Carlson calculated the free energy available to the sperm to be no more than four times the energy needed hydrodynamically for motility. As Bishop [I] has pointed out, however, this does not take into account the variation in oxygen consumption by the sperm during the course of the experiment. Taking this variation into account means that the available energy may exceed the hydrodynamic dissipation by as much as 20 times at the start of the experiment. Ai relation between energy production and motility in sperm can only be demonstrated clearly when during the experiment a large variation in metabolism and motility can be induced. In order to obtain this, the preparation used should have only one metabolic pathway going (for example the respiratory chain, under exclusion of fructolysis and glycolysis), which can be readily inhibited. Xmytal sodium seems a very suitable inhibition for this purpose. From spectrophotometric measurements, Gonse [6] has established that the action of amytal on sperm is the same as on mitochondria preparations of other tissues such as heart muscle or liver. Respiration is blocked at the level of the flavo-proteins, and there is no apparent uncoupling action. Hence, amytal may be assumed to al‘fect equally all phosphorylation sites of sperm. IN
Experimental
Cell
Xesearch
37
Respiration
and motility
of spermatozoa
313
The experiments reported in this paper are concerned with (1) measurement of steady-state values of respiration and motility of bull spermatozoa before and after stepwise inhibition with amytal; (2) with the kinetics of the action of amytal. A kinetic study is necessary to establish that the membrane of the sperm is sufficiently permeable to amytal. Also, an observed delay between the readjustment of the respiration and that of the motility to the inhibited level allows one to make an estimate of energy reserves stored in spermatozoa. MATERIALS
AND
EXPERIMENTAL
METHODS
Sperm sourceand handling.-Bull spermatozoa were kindly provided by the New York Artificial Breeders Cooperative in Ithaca, N.Y. After collection, 1 ml of semen was diluted 5 times in a modified Krebs Ringer Phosphate buffer which contained 10 per cent egg yolk (hereafter called EB). The semen was flown into New York City while cooling down to 4°C. Experiments were completed 8 to 14 hr after collection of the semen. For the experiments to be described the semenhas to be washed free of substrates present in the seminal plasma. It proved impossible to perform washing of sperm in pure mineral solutions while preserving a good motility afterwards. If washing was done with the EB, however, motility after washing was on visual estimation not noticeably reduced, and movement of the sperm was of the rotating, normal type [18]. Washing consisted of spinning down the 5 x diluted semen for 8 min at 400 g, replacing supernatant with EB, followed by 6 min centrifugation at 400 g, and a secondreplacement of supernatant with EB. 0.2 to 0.6 ml of the washed sperm were diluted in EB in the measuring chamber to a total volume of 2 ml, and a concentration of 0.6 to 1.5 y 10’ normally moving sperm/ml. 10 mM sodium pyruvate was added as substrate for the sperm. Agglutination was avoided by adding 40 ~1 of seminal plasma (2 per cent of the volume) into the measuring chamber. The seminal plasma had been thoroughly dialyzed against 0.154 M NaCl and filtered to make it optically clear. In this procedure the factor described by Lindahl [9] which prevents sperm agglutination is obviously retained. The amount of fructose and glucose present in the final suspensionwas ~2 pig/ml (Nelson-Somogyi determination [ll, 231). The percentage of moving sperm in the samplesreceived varied between 10 and 70 per cent. Only sperm sampleswhich had at least 50 per cent moving cells as determined by visual estimation upon arrival were used for the experiments. Preparation of the diluent fluid.-A modified Krebs Ringer Phosphate buffer containing the equivalent of 10 per cent egg yolk (EB) was prepared as follows: Egg yolk from freshly laid eggs, carefully separated from the egg white and the yolk membrane, was diluted with 2 volumes of 0.154 M NaCl. 1000 1.U. of penicillin and 1000 pg of streptomycin were added per ml of the mixture. The diluted egg yolk was centrifuged at 100,000 g for 90 min. The bulk of the lipo-proteins of the egg yolk is sedimented this way [22]. The clear-yellow supernatant of the centrifugation Experimenfal
Cell Research
37
314
R. Rikmenspoel
was dialysed 4 times at 4°C against 0.154 M NaCl. In this way all heavy metal ions and all small molecules which could serve as substrate for the sperm are removed. After dialysis the egg-yolk containing fluid was diluted so that the final solution contained 0.144 M NaCl, 0.006 M KCl, 0.002 M Mg SO,, 0.002 M CaCl, 10 per cent 0.1 M phosphate buffer and the equivalent of 10 per cent egg yolk. The pH of the solution was 7.2. The solution was filtered through a 0.3 ,Upore width Millipore filter to make it sterile and optically clear [15] and sealed in vials of IO ml each. The sealed vials were heated twice for 30 min at 56°C in order to destroy any respiratory activity of the fluid. The described fluid contained i 1 ,ug/ml oxidizable sugars (Nelson-Somogyi determination). The respiratory activity as measuredwith the oxygen electrode was < 0.3 ,oMO,/min at 37°C. Measurement techniques.-The measuring chamber in which the experiments were performed is shown schematically in Fig. 1. The chamber consists of a 2 ml cuvet which contains the bulk sample. Oxygen concentration of the suspensionis recorded by means of a Clark oxygen electrode.1 Additions to the cuvet can be made with a Hamilton syringe, the needle of which fits snugly in an opening of the cuvet cover. Stirring of the suspensionis done by a small magnetic stirrer. A slide having a channel of 7 mm wide and 30 p deep is connected through a slot to the cuvet. Observation of the sperm motility is made by a microscope which views the channel in the slide. After an addition has been made in the cuvet, the samplein the slide can be replaced by an aliquot of the bulk sample in the new condition. This is done by adding some reaction medium to the cuvet by a secondHamilton syringe, while opening the rubber stopper which otherwise sealsoff the “open” end of the slide. A full description of the chamber is given elsewhere [13]. By means of a photoelectric and electronic method [12, 14, 161 the number of sperm which move normally, and their average velocity were measured. These measurementsare made with an accuracy of 115 per cent (s.D.) for the number of sperm, and of +7 per cent (SD.) for the average velocity. In those circumstances where the concentration of moving sperm dropped below 0.3 x IO7 ml, or the average velocity below 50 ,u/secphotographs of the sampleswere taken at an exposure time of 1 sec. Moving sperm produce “tracks” on the negative. By measuring the average length of the tracks the average velocity of the sperm is obtained. By counting the number of tracks per cm2 the concentration is obtained. This procedure, introduced by Rothschild [20], was worked out in detail earlier [16]. Motility measurements concerned with the kinetics of amytal action, described in section 3.3, were all made with this photographic method. All experiments were performed at a sample temperature of 35.0 10.2% (see ref. [131). Amytal
sodium.-Amytal sodium was obtained from Eli Lilly Company in ampules of 125 mg each. Purity of the amytal, according to U.S.P., was better than 98.5 per cent. For each experiment amytal was freshly dissolved to a 0.1 M and 0.5 M solution. 1 Yellow Experimental
Spring
Instrument
Cell Reseurch
Company, 37
Yellow
Springs,
Ohio.
Respiration
and motility
of spermatozoa
315
RESULTS
Control experiments.--Sperm samples, incubated in the measuring chamber at 35°C as described on p. 313, show a respiration and motility only slightly decreasing with time over a period of 30 to 40 min. Of a typical experiment
Fig. l.-Top view (cover of cuvet removed) and side view the micro chamber used in the periments. The insert shows connection between the slide the cuvet. (Courtesy of Rev. Znsfr.)
the of exthe and Sci
o I
the tracing recorded by the oxygen electrode is shown in Fig. 2. In Fig. 2 are also inserted the values for number of moving cells/ml and for the average velocity of the moving cells as measured during the course of the experiment. The slide on which the motility observations were made was “flushed” just prior to each motility measurement. Fig. 3 shows the value of respiration and motility as a function of time for four different sperm samples. The value of the respiration is plotted as the fraction of the respiration during the period the first motility measurement was made. In order to combine all concentration measurements into one graph, the 21-651810
Experimental
Cell Research
37
316 following measured was drawn were then nate. This
R. Rikmenspoel procedure of reducing the data was followed: for each sample the concentration c was plotted as a function of time. A smooth line which fitted visually best between the measured points. The data plotted relative to the point, cO, where this line intersected the ordiprocess, illustrated in Fig. 4, avoids an accumulation of the errors
Time
(minutesI
Fig.
Tbme
2.
(min.1
-
Fig. 3.
Fig. 2.-Tracing of the oxygen electrode obtained from a sperm sample incubated at 35°C. The values for the average velocity of the sperm and for the number of moving sperm, measured at three moments during the incubation are inserted in the figure. The tracing was interrupted when the slide was flushed prior to a motility measurement. Fig. 3. Change samples.
of respiration
and
motility
during
incubation
at 35’C
of four
different
sperm
in the measurement of c, which are rather large, and avoids also giving a false emphasis of accuracy on the first concentration measurement taken of a sample. The average velocities, B, are plotted without corrections. It can be observed from Fig. 3, that the decay of the number of moving cells c is fastest and therefore, sets a limit to the maximal permissible duration of Experimental
Cell
Research
37
Respiration
and motility
317
of spermatozoa
an experiment. The decay in c amounts to about 12 per cent in 20 min and about 25 per cent in 35 min. In doing the experiments on amytal inhibition care was taken always to finish the experiments in about 25 min after warming up of the sample. This is sufficient to take 4 to 5 “steps” on each sample. As the accuracy of
:0
---_
---_
--__
0
0 --W_
--__
0
I
I
I
I
IO
20
30
40
Time
Fig. Fig. $.-Diagram cells.
--__ 0
(min.)
4.
illustrating
the method
Fig. B.-Tracing of the oxygen electrode interrupted, and the zero of the recorder and the slide was flushed. The measured inserted in the figure.
of reducing
the data
found
for the number
of moving
during a typical inhibition experiment. The tracing was was readjusted when an addition of amytal was made values for the motility of the sperm at each step are
the data is not very high, especially in the later parts of an inhibition experiment where motility and respiration are strongly reduced, no correction was made towards the decay of the sample during the experiment. Quantitative description of amytal inhibition.-When amytal is added to a sperm preparation the respiration and motility appear to be reduced instantly, as has been reported by Gonse [6]. Through the experiments described in the 3rd part of this section, it was established that the new steady state is reached in about one minute after the addition of the amytal. Hence, in the Experimental
Cell
Research
37
318
R. Rikmenspoel
experiments discussed in this section, the procedure followed was to wait one minute after an addition was made in the cuvet, before the sample in the slide was changed and the motility measurement was started. A typical experiment is illustrated in Fig. 5, which shows the recording obtained with the oxygen electrode, first with only EB in the cuvet. After
Per cent
--L-L-0
2
4
Amytal
6
8
IO mM
0
concentration
Fig.
2
4
Amytol
6.
6
Fig.
Fig. 6.-Inhibition, measured on ten samples, of respiration shows the mean values obtained by averaging the points 1 mM, 2mM, 3 m&f, 5 mM and 10 mM respectively. Fig. 7.-Change in the number of moving sperm for of amytal concentration. The insert shows the mean at an amytal concentration of 0 mM, 1 mM, 2 mM, 3 7, 8, 9 an identical symbol is used for the same sperm
8
concentration
7.
of bull sperm by amytal. at an amytal concentration
The insert of 0 m&f,
10 different sperm samples, as a function values obtained by averaging the points mM, 5 mM, 10 m&f respectively. In Figs. sample.
the sperm suspension has been added in the cuvet, a constant respiration of 4.2 PMOJmin is observed. The subsequent addition of 0.8 mM, 1.2 m&I, 3 mM amytal results in reducing the respiration to 1.8 ,uMO,/min, 1.2 pMO,/min, 0.5 ,uMO,/min respectively. The data obtained for the motility in each of the inhibition steps are inserted in Fig. 5. Both the average velocity, B, and the number of sperm, c, which are moving, are shown decreasing with the higher amytal concentration. Even though the photoelectric method used for measuring motility, does not allow quantitative determination of the halfwidth of the velocity distribution [14], it still indicates qualitatively the shape of this distribution. It was observed that at each of the “inhibition steps”, a “bell shaped” velocity Experimental
Cell Research
37
Respiration
and motility
of spermatozoa
319
distribution is present. The consequences of this observation are discussed on p. 323. The results of 10 different experiments were consistent enough to allow them to be pooled. Fig. 6 shows the effect of amytal on the respiration of the 10 samples. The respiration is plotted as the fraction of the value for the
0
2
4
6
8
IOmM
Fig. 8.-Change in average velocity in ten different sperm samples, as a function of the amytal concentration. Amytal
concentration
uninhibited preparation. Fifty per cent inhibition is obtained at an amytal concentration of approximately 1.2 mM. Fig. 7 represents the effect of amytal on the number of moving sperm. The same correction procedure as described on pp. 316-317 (Fig. 4) was applied here. It can be seen that when 90 per cent inhibition of the respiration is obtained, at around 10 mM amytal, only about 25 per cent of the number of original cells are still moving. This curious effect is referred to in more detail in the discussion. Fig. 8 shows the decrease of the average velocity with increasing amytal concentration. As the initial velocity does not show a big spread for the different samples the data are plotted absolutely. In Fig. 9 all data points have been pooled together and plotted as respiration in pMO,/min versus number of moving cells X average velocity. All data have been presented here by their absolute value, without any of the previously described correction applied. Fig. 9 shows that in the sperm system used the “amount” of motility, expressed as (cu) is in direct relation to the respiration. The relation holds over a range of respiration values of 10 ,&lO,/min to 0.3 pMO,/min, and of “motility” values from 240 to 6 X 10’ Experimental
Cell Research
37
320
R. Rikmenspoel
sperm ,u/sec. This represents roughly a range of a factor of 30. This can be considered as evidence that the amytal has no “poisoning” effect but only restricts itself to inhibition of the respiratory chain with a consequent reduction of production of free energy available to the sperm. The straight line drawn in Fig. 9 which best fits the measured points leads to a relation where
Fig. 9.-Respiration of ten different sperm samples plotted versus the “amount” of motility, expressed as number of moving sperm x average velocity. The variation within the samples is obtained by stepwise inhibition by amytal (cf. Fig. 5).
c / o,,LLLLI_LLL_I
Lllll’
I I III”!
IO
Concentrolion
102
in lO’/ml
x average
I03
velocity
in p/set.
respiration is proportional to (c X u)p, p being 0.95. In view of the large scattering, the data of Fig. 9 cannot be considered inconsistent with p = 1. If the respiration R is expressed in mol O,/sec, c in number of sperm, and v in cm/set, the relation can be written as R = a(c x v)molO,/sec
(1)
CChaving the value of a = 6.1 X lo-16 mol Oz/sec/sperm/cm/sec. Kinetics of amytal action.-Determination of the speed with which amytal takes effect is important because it gives information about the permeability of the membrane of the sperm. By the delay between the decrease in respiration and that in the motility, it sets a limit to the amount of energy rich bonds (,P) which are stored in the sperm. The measuring chamber described is not suitable for measurement of fast changes in respiration. When additions are made in the cuvet, or when the slide is flushed, violent irregular signals occur which overload the amplifier used. Therefore, for measuring the speed of change of respiration Experimental
Cell Research
37
Respiration
and motility
321
of spermatozoa
upon addition of amytal, the oxygen electrode was operated in a separate cylindrical glass cuvet, as described by Chappell [5]. It was found that in these conditions the indication time of the electrode was the limiting factor in determining the speed of action of amytal. This is illustrated in Fig. 10, which shows that a new steady state of respiration is indicated by the electrode
I
Amytal IO mM
1
‘~‘402
*
Fig. lO.-Tracing of the oxygen electrode during the addition of amytal. The sensitivity and the time-scale are greatly expanded compared to those in Figs. 2 and 5. The jump in oxygen level represents the amount of oxygen dissolved in the amytal solution.
: I
; ’
it
60 sec.
*
within 10 set after the addition. In 4 experiments performed the results were identical; the new steady state is reached within the indication time of the oxygen electrode. In order to obtain information about the permeability of the membrane, an indication time of at least 10 x faster than now available would be required. The kinetics of the motility change upon amytal inhibition was studied using the micro chamber and the photographic “track” technique. The I. Delay z between the addition of amytal and the moment readjustment to the inhibited steady state has occurred.
TABLE
Amytal addition m&f
7 initial Wet)
v final Wsec)
t (set)
1 2 3
1.5
78 74 80
26 27 24
88 70 46
4 5 6
10
92 98 80
55 52 45
45 53 70
Experiment No.
Experimental
that 90 %
Average of t (set)
68
56 1 Cell Research
37
322
R. Rikmenspoel
procedure was as follows: after the sample in the chamber was warmed up to the point where constant motility was reached, the camera was put in place and focussed. The addition of amytal was made with one of the Hamilton syringes. Three seconds were allowed for stirring of the content of the cuvet, and then the slide was flushed by means of the second syringe.
Amytal 110
t
Seconds
Fig. Fig. Il.-Change amytal.
with
time
Fig.
11. of the average
velocity
of a sperm
sample
upon
12. addition
of 1.5 mM
Fig. 12.-Schematic form of the function g(u). The peak at v = o represents the cells which have become motionless upon addition of amytal. The cross-hatched area represents the fraction of the cells, having an original velocity u,,, which is still moving after the amytal addition. The dotted peak at u,, shows the cells before the amytal addition.
Photographs of the slide were made at 5 set interval, starting 10 set after the amytal addition. One minute after the addition the interval between photographs was increased to 10 sec. At each photograph 6 to 14 tracks were recorded from moving sperm. This is sufficient to define the average velocity u of the sperm at that moment to about +lO p/set. It was not considered that figures about the average number of moving sperm can be deduced from so few recorded tracks, so only the decrease of u with time is reported. Two groups of 3 experiments each were performed. The first group was aimed at approximately half inhibition by the addition of 1.5 mM, and the Experimental
Cell Research
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Respiration
and motility
of spermatozoa
323
second group at maximal inhibition by addition of 10 mM amytal. As the contents of the cuvet were probably not completely mixed during the 3 set allowed for stirring, the actually measured inhibition of v should not be read as being quantitatively related to the amytal concentration used. Fig. 11 shows the results of a partial-inhibition experiment. Table I summarizes the results of the 6 experiments performed. It can be seen that the new, inhibited, steady state is reached approximately 1 min after the addition. There is no apparent difference between the two groups.
DISCUSSION
The action of amytal.-The data presented in Figs. 6, 7 and 8 show that the action of amytal on sperm motility is a complicated one. As mentioned before, it cannot be assumed that the addition of a certain concentration of amytal changes the original velocity v, of a sperm into v = tlvO, tc being the same for all sperm. In that case no decrease in the number of moving cells c should be found, as the velocity distribution simply would shift towards the ordinate. In general we can say that a sperm having originally a velocity v, will have a chance g(v) of moving with a velocity v after the addition of amytal. Qualitatively one can say about the function g(v): 1. For v = 0, which represents sperm that have become motionless, g(v) has a value +O. So Jg(v)d v, excluding the region near v = 0, should have a value < 1. 2. As the velocity distribution after the addition of amytal still has a “bell shape”, with small values for the lower velocities, it follows that the function g(v) has to be small for the lower velocities also. Obviously g(v) also has a “bell shape”, qualitatively sketched in Fig. 12. The three important quantities of g(v) are: the fraction A of cells which still move, represented by the cross-hatched area in Fig. 12, the mean, B, of g(v) and its half width y. A, B and y are of course functions of the original velocity v,. If the form of the velocity distribution of the sperm before and after addition of amytal is known, it is possible to solve A, B, and y in first order dependence on v,. Using the photographic method, experiments to measure the form of the velocity distributions are presently being undertaken, with the aim of obtaining quantitative information on A, B and y. Energy considerations.-The data presented in Fig. 9 and on p. 320 can be regarded as strong evidence that the motility is directly dependent on the steady state level of ATP production. If the coupling of the oxidative Experimenlal
Cell
Research
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R. Rikmenspoel
324
phosphorylation were maximal, at a P/O ratio = 3, the maximal amount of free energy, F, available to the sperm in our preparation can be calculated from eq (l), p. 320, assuming 9 kcal to be liberated at hydrolysis of 1 mol ATP [S]: F = 1.4 X 10-S (c x u) erg/set. (2) with c being the number of moving cells, and u the average velocity in cm/set. Taylor [24] has estimated the amount of work performed hydrodynamically by a moving flagella. Using as a model for the flagella a thin rod in which flat sinusoidal, flexural, waves are traveling, Taylor finds W = 4n3,uf2b2L/ (0.62-ln(ne/l) where: p = viscosity of the fluid; f = frequency of the tail wave; b = amplitude of the tail wave; 1 = wavelength of the tail wave; L = length of the tail; Q = diameter of the tail. The sperm observed in our experiments, however, show a three-dimensional tail wave. It has been shown recently [17] that the two components of the helical wave have amplitudes which have a ratio of 2 to 1. In the first order approximation used by Taylor the work performed by the two components of the helical wave can be taken to be linearly additive [24]. This yields for the hydrodynamic dissipation of the sperm studied in this paper: W=-
6 n3/xf2bzL 0.62 - ln(ne/il)
Carlson [3], using a more complicated model for the flagella, arrived at essentially the same results as Taylor. The following values have been adopted for some of the quantities appearing in eq (3): p = 1.0 X 1O-2 poise [16]; L = 60 ,u; 3, = 35 ,u [14]; Q = 0.5 ,L For rotating cells u has been found previously [ 121 to be: v=eb2, being E = 1.2 X 104/cm/set. Data on fare not as well defined. For non-rotating bull sperm it is reported [14] that f * 22 cps, with no clear relation between f and v. Gray has reported lower values [7] for f, f = 10 cps in non-rotating bull sperm. It has been shown recently, however, that this is due to insufficient time resolution of the cinemicrographs from which these data were obtained. From cinemicrographs with a time resolution of 5 msec it was found by the author that for 10 rotating cells the frequency of the tailwave was between 20 and 24 cps [17]. For these 10 cells no correlation between f and v was found. As an average E
Experimental
Cell Research
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Respiration
and motility
of spermalozocr
325
figure is used here: f = 22 cps. It should be realized, however, that some uncertainty exists as to the correctness of the mean value for /as used here. If all these values are inserted in eq (3) we find that the work \\’ performed by a number c of sperm is \V= 2.25 X 1O-4 (c X II) erg/set. The energy dissipated in the elastic deformation of the tail has to be taken into account. Jlachin [lo] has calculated that the amount of work needed for elastic deformation of the tail is :f- of the hydrodynamic dissipation. This brings the total work \V tOt performed by moving sperm to
nyt,,= 3.0 x lO-4
(cx 0) crg/sec.
(4)
A comparison of equation (2) and (4) indicates that the “efficiency” of the oxidative metabolism is at least about 22 per cent. This figure is arrived at by assuming the oxidative phosphorylation to be tightly coupled. Chance [4] has pointed out, however, that under conditions of less tight coupling the thermodynamic efficiency may be lowered, but the amount of ATP produced for a ccl1 may actually be higher, due to the much faster turnover. In view of the wide range over which the apparent efficiency reported here seems to be constant, one would assume that under our conditions the coupling has remained fairly constant. Actually measured P/O ratio’s in sperm should, therefore, be of great interest. Energy reseraes in sperm.-The results of the esperiments on the kinetics of amytal action as illustrated in Fig. 10 show that the electron flo\v in the respiratory chain is readjusted within 10 set after the addition of amytal. The production of ATI’ is readjusted at Lhe same time to its new level. The slojver decay of the motility (Fig. 11) can be seen as an exhausting of reserves of ATI’ available in the sperm. From the data of Fig. 11 and Table I it can be very roughly concluded that stored energy in our sperm preparation is sufficient to maintain motility over a period of the order of 30 sec. According to cq (4) the amount of work performed by one normally moving sperm in that period \\ould be w 10-O erg. Hornstein and Steberl ;2: have reported \:alues of =\‘l’P content of bull semen of approximately lo-13 g ATP/sperm. The free energy obtainable by hydrolysis of this amount would come to 0.8 x 1V4 erg/sperm. A comparison of this figure \vith the amount of \vork performed as mentioned above (w 1W4 erg/sperm) is again a confirmation that the motility of sperm is directly dctcrminrd by the instantaneous amount of ATP made available to the sperm. Experimenld
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326
SUMMARY
Simultaneous measurements of motility and respiration of spermatozoa have been performed using a specially developed microchamber. Of each sperm sample used the respiration and the motility was stepwise reduced in the course of the experiment by additions of amytal. Kinetic studies showed that after the addition of amytal the new steady state level of respiration is reached within 10 set and that of the motility in about 60 sec. Steady state values of respiration plotted versus the steady state values of number of moving sperm X average velocity show an almost linear relation, which holds over a range of a factor 30. From this relation can be deduced that the amount of energy needed for the sperm motility is at least 22 per cent of the free energy which can be produced by the oxidative metabolism. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BISHOP, D. W., Physiol. Rev. 42, 1 (1962). BOMSTEIN, R. A. and STEBERL, E. A., Expfl Cell Res. 18, 217 (1959). CARLSON, F. D., Proc. 1st. Nat. Biophys. Conf. Yale Un. Press, 1959. CHANCE, B. and WILLIAMS, G. R.. Adu. Enzumol. 17. 65 (1956). CHAP&L, B., in T. W. Gobnwr~‘and O.Lr~“o~mm (ids.): Biological Structure and Function. Academic Press, New York, 1961. GONSE, P., in D. W. BISHOI~ (ed.), Spermatozoan Motility. AAAS, Washington, D.C., 1962. GRAY, J., J. Exptl Biol. 35, 96 (1958). LEHNINGER, A. L., Rev. Mod. Phys. 31, 136 (1959). LINDAHL, P. E. and BRATTSAND, R.. Zool. Bidraa. Uam-ala 35. 505 11962). MACHIN,‘K. E., J. Exptl Biof. 35, 796 (1958). ” ” NELSON, N., J. Biol. Chem. 153, 375 (1944). RIKMENSPOEL, R., in D. W. BISHOP (ed.), Spermatozoan Motility. AAAS, Washington, D.C., 1962. __ Reu. Sci. Instr. 35, 49 (1964). __ ibid. 35, 52 (1964). ~ Experienfia 13, 124 (1957). __ Photoelectric and Cinematographic Observations of the Motility of Bull Sperm Cells. Thesis, J. A. Smit, Utrecht, 1957. ~ Trans. N.Y. Acad. Sci., Series II 26, no. 8 Suppl. (1964). In press. RIKMENSPOEL, R., VAN HERPEN, G. and EIJKHOUT, P., Phys. in Med. and Biol. 5,167 (1960). ROTHSCHILD, LORD, Proc. Roy. Sot. B 151, 1 (1959). __ in G. E. W. WOLSTENHOLME, M. P. CAMERON and J. S. FREEMAN (eds.), Mammalian Germ Cells. J. &. A. Churchill, London, 1953. ROTHSCHILD, LORD and CLELAND, K. W., J. Ezpfl Biol. 29, 66 (1952). SHEPARD, C. S. and MOTTLE, G. A., .I. Biol. Chem. 179, 369 (1949). SOMOGYI, M., J. Biol. Chem. 160, 61 (1945). TAYLOR, G. I., Proc. Roy. Sot. A 211, 225 (1952).
Experimental
Cell Research
37
Experimental
THE
Cell Research
37, 312-326
(1965)
INHIBITION BY AMYTAL OF RESPIRATION AND MOTILITY OF BULL SPERMATOZOA R.
The Population
Council,
RIKMENSPOEL
The Rockefeller Received
March
Institute,
New
York,
N.Y.,
U.S.A.
16, 1964
recent papers [ 13, 14, 161 apparatus has been described which makes it possible to simultaneously measure, on the same preparation, motility and respiration of sperm suspensions. This system has been developed to investigate relations between the oxidative metabolic pathway as an energy producer for sperm and their motility as an energy consumer. These relations cannot a priori be assumed to exist. Calorimetric measurements by Rothschild [19] of the heat production by anaerobic metabolism of bull sperm gave values of 6 to 9 X lo-6 erg/sperm/set. This is more than one order of magnitude more than estimates by Carlson [3] of the energy which is dissipated by the moving flagella in the surrounding fluid. From data of Rothschild and Cleland [21] on the oxygen consumption of sea urchin sperm, Carlson calculated the free energy available to the sperm to be no more than four times the energy needed hydrodynamically for motility. As Bishop [I] has pointed out, however, this does not take into account the variation in oxygen consumption by the sperm during the course of the experiment. Taking this variation into account means that the available energy may exceed the hydrodynamic dissipation by as much as 20 times at the start of the experiment. Ai relation between energy production and motility in sperm can only be demonstrated clearly when during the experiment a large variation in metabolism and motility can be induced. In order to obtain this, the preparation used should have only one metabolic pathway going (for example the respiratory chain, under exclusion of fructolysis and glycolysis), which can be readily inhibited. Xmytal sodium seems a very suitable inhibition for this purpose. From spectrophotometric measurements, Gonse [6] has established that the action of amytal on sperm is the same as on mitochondria preparations of other tissues such as heart muscle or liver. Respiration is blocked at the level of the flavo-proteins, and there is no apparent uncoupling action. Hence, amytal may be assumed to al‘fect equally all phosphorylation sites of sperm. IN
Experimental
Cell
Xesearch
37
Respiration
and motility
of spermatozoa
313
The experiments reported in this paper are concerned with (1) measurement of steady-state values of respiration and motility of bull spermatozoa before and after stepwise inhibition with amytal; (2) with the kinetics of the action of amytal. A kinetic study is necessary to establish that the membrane of the sperm is sufficiently permeable to amytal. Also, an observed delay between the readjustment of the respiration and that of the motility to the inhibited level allows one to make an estimate of energy reserves stored in spermatozoa. MATERIALS
AND
EXPERIMENTAL
METHODS
Sperm sourceand handling.-Bull spermatozoa were kindly provided by the New York Artificial Breeders Cooperative in Ithaca, N.Y. After collection, 1 ml of semen was diluted 5 times in a modified Krebs Ringer Phosphate buffer which contained 10 per cent egg yolk (hereafter called EB). The semen was flown into New York City while cooling down to 4°C. Experiments were completed 8 to 14 hr after collection of the semen. For the experiments to be described the semenhas to be washed free of substrates present in the seminal plasma. It proved impossible to perform washing of sperm in pure mineral solutions while preserving a good motility afterwards. If washing was done with the EB, however, motility after washing was on visual estimation not noticeably reduced, and movement of the sperm was of the rotating, normal type [18]. Washing consisted of spinning down the 5 x diluted semen for 8 min at 400 g, replacing supernatant with EB, followed by 6 min centrifugation at 400 g, and a secondreplacement of supernatant with EB. 0.2 to 0.6 ml of the washed sperm were diluted in EB in the measuring chamber to a total volume of 2 ml, and a concentration of 0.6 to 1.5 y 10’ normally moving sperm/ml. 10 mM sodium pyruvate was added as substrate for the sperm. Agglutination was avoided by adding 40 ~1 of seminal plasma (2 per cent of the volume) into the measuring chamber. The seminal plasma had been thoroughly dialyzed against 0.154 M NaCl and filtered to make it optically clear. In this procedure the factor described by Lindahl [9] which prevents sperm agglutination is obviously retained. The amount of fructose and glucose present in the final suspensionwas ~2 pig/ml (Nelson-Somogyi determination [ll, 231). The percentage of moving sperm in the samplesreceived varied between 10 and 70 per cent. Only sperm sampleswhich had at least 50 per cent moving cells as determined by visual estimation upon arrival were used for the experiments. Preparation of the diluent fluid.-A modified Krebs Ringer Phosphate buffer containing the equivalent of 10 per cent egg yolk (EB) was prepared as follows: Egg yolk from freshly laid eggs, carefully separated from the egg white and the yolk membrane, was diluted with 2 volumes of 0.154 M NaCl. 1000 1.U. of penicillin and 1000 pg of streptomycin were added per ml of the mixture. The diluted egg yolk was centrifuged at 100,000 g for 90 min. The bulk of the lipo-proteins of the egg yolk is sedimented this way [22]. The clear-yellow supernatant of the centrifugation Experimenfal
Cell Research
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314
R. Rikmenspoel
was dialysed 4 times at 4°C against 0.154 M NaCl. In this way all heavy metal ions and all small molecules which could serve as substrate for the sperm are removed. After dialysis the egg-yolk containing fluid was diluted so that the final solution contained 0.144 M NaCl, 0.006 M KCl, 0.002 M Mg SO,, 0.002 M CaCl, 10 per cent 0.1 M phosphate buffer and the equivalent of 10 per cent egg yolk. The pH of the solution was 7.2. The solution was filtered through a 0.3 ,Upore width Millipore filter to make it sterile and optically clear [15] and sealed in vials of IO ml each. The sealed vials were heated twice for 30 min at 56°C in order to destroy any respiratory activity of the fluid. The described fluid contained i 1 ,ug/ml oxidizable sugars (Nelson-Somogyi determination). The respiratory activity as measuredwith the oxygen electrode was < 0.3 ,oMO,/min at 37°C. Measurement techniques.-The measuring chamber in which the experiments were performed is shown schematically in Fig. 1. The chamber consists of a 2 ml cuvet which contains the bulk sample. Oxygen concentration of the suspensionis recorded by means of a Clark oxygen electrode.1 Additions to the cuvet can be made with a Hamilton syringe, the needle of which fits snugly in an opening of the cuvet cover. Stirring of the suspensionis done by a small magnetic stirrer. A slide having a channel of 7 mm wide and 30 p deep is connected through a slot to the cuvet. Observation of the sperm motility is made by a microscope which views the channel in the slide. After an addition has been made in the cuvet, the samplein the slide can be replaced by an aliquot of the bulk sample in the new condition. This is done by adding some reaction medium to the cuvet by a secondHamilton syringe, while opening the rubber stopper which otherwise sealsoff the “open” end of the slide. A full description of the chamber is given elsewhere [13]. By means of a photoelectric and electronic method [12, 14, 161 the number of sperm which move normally, and their average velocity were measured. These measurementsare made with an accuracy of 115 per cent (s.D.) for the number of sperm, and of +7 per cent (SD.) for the average velocity. In those circumstances where the concentration of moving sperm dropped below 0.3 x IO7 ml, or the average velocity below 50 ,u/secphotographs of the sampleswere taken at an exposure time of 1 sec. Moving sperm produce “tracks” on the negative. By measuring the average length of the tracks the average velocity of the sperm is obtained. By counting the number of tracks per cm2 the concentration is obtained. This procedure, introduced by Rothschild [20], was worked out in detail earlier [16]. Motility measurements concerned with the kinetics of amytal action, described in section 3.3, were all made with this photographic method. All experiments were performed at a sample temperature of 35.0 10.2% (see ref. [131). Amytal
sodium.-Amytal sodium was obtained from Eli Lilly Company in ampules of 125 mg each. Purity of the amytal, according to U.S.P., was better than 98.5 per cent. For each experiment amytal was freshly dissolved to a 0.1 M and 0.5 M solution. 1 Yellow Experimental
Spring
Instrument
Cell Reseurch
Company, 37
Yellow
Springs,
Ohio.
Respiration
and motility
of spermatozoa
315
RESULTS
Control experiments.--Sperm samples, incubated in the measuring chamber at 35°C as described on p. 313, show a respiration and motility only slightly decreasing with time over a period of 30 to 40 min. Of a typical experiment
Fig. l.-Top view (cover of cuvet removed) and side view the micro chamber used in the periments. The insert shows connection between the slide the cuvet. (Courtesy of Rev. Znsfr.)
the of exthe and Sci
o I
the tracing recorded by the oxygen electrode is shown in Fig. 2. In Fig. 2 are also inserted the values for number of moving cells/ml and for the average velocity of the moving cells as measured during the course of the experiment. The slide on which the motility observations were made was “flushed” just prior to each motility measurement. Fig. 3 shows the value of respiration and motility as a function of time for four different sperm samples. The value of the respiration is plotted as the fraction of the respiration during the period the first motility measurement was made. In order to combine all concentration measurements into one graph, the 21-651810
Experimental
Cell Research
37
316 following measured was drawn were then nate. This
R. Rikmenspoel procedure of reducing the data was followed: for each sample the concentration c was plotted as a function of time. A smooth line which fitted visually best between the measured points. The data plotted relative to the point, cO, where this line intersected the ordiprocess, illustrated in Fig. 4, avoids an accumulation of the errors
Time
(minutesI
Fig.
Tbme
2.
(min.1
-
Fig. 3.
Fig. 2.-Tracing of the oxygen electrode obtained from a sperm sample incubated at 35°C. The values for the average velocity of the sperm and for the number of moving sperm, measured at three moments during the incubation are inserted in the figure. The tracing was interrupted when the slide was flushed prior to a motility measurement. Fig. 3. Change samples.
of respiration
and
motility
during
incubation
at 35’C
of four
different
sperm
in the measurement of c, which are rather large, and avoids also giving a false emphasis of accuracy on the first concentration measurement taken of a sample. The average velocities, B, are plotted without corrections. It can be observed from Fig. 3, that the decay of the number of moving cells c is fastest and therefore, sets a limit to the maximal permissible duration of Experimental
Cell
Research
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Respiration
and motility
317
of spermatozoa
an experiment. The decay in c amounts to about 12 per cent in 20 min and about 25 per cent in 35 min. In doing the experiments on amytal inhibition care was taken always to finish the experiments in about 25 min after warming up of the sample. This is sufficient to take 4 to 5 “steps” on each sample. As the accuracy of
:0
---_
---_
--__
0
0 --W_
--__
0
I
I
I
I
IO
20
30
40
Time
Fig. Fig. $.-Diagram cells.
--__ 0
(min.)
4.
illustrating
the method
Fig. B.-Tracing of the oxygen electrode interrupted, and the zero of the recorder and the slide was flushed. The measured inserted in the figure.
of reducing
the data
found
for the number
of moving
during a typical inhibition experiment. The tracing was was readjusted when an addition of amytal was made values for the motility of the sperm at each step are
the data is not very high, especially in the later parts of an inhibition experiment where motility and respiration are strongly reduced, no correction was made towards the decay of the sample during the experiment. Quantitative description of amytal inhibition.-When amytal is added to a sperm preparation the respiration and motility appear to be reduced instantly, as has been reported by Gonse [6]. Through the experiments described in the 3rd part of this section, it was established that the new steady state is reached in about one minute after the addition of the amytal. Hence, in the Experimental
Cell
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37
318
R. Rikmenspoel
experiments discussed in this section, the procedure followed was to wait one minute after an addition was made in the cuvet, before the sample in the slide was changed and the motility measurement was started. A typical experiment is illustrated in Fig. 5, which shows the recording obtained with the oxygen electrode, first with only EB in the cuvet. After
Per cent
--L-L-0
2
4
Amytal
6
8
IO mM
0
concentration
Fig.
2
4
Amytol
6.
6
Fig.
Fig. 6.-Inhibition, measured on ten samples, of respiration shows the mean values obtained by averaging the points 1 mM, 2mM, 3 m&f, 5 mM and 10 mM respectively. Fig. 7.-Change in the number of moving sperm for of amytal concentration. The insert shows the mean at an amytal concentration of 0 mM, 1 mM, 2 mM, 3 7, 8, 9 an identical symbol is used for the same sperm
8
concentration
7.
of bull sperm by amytal. at an amytal concentration
The insert of 0 m&f,
10 different sperm samples, as a function values obtained by averaging the points mM, 5 mM, 10 m&f respectively. In Figs. sample.
the sperm suspension has been added in the cuvet, a constant respiration of 4.2 PMOJmin is observed. The subsequent addition of 0.8 mM, 1.2 m&I, 3 mM amytal results in reducing the respiration to 1.8 ,uMO,/min, 1.2 pMO,/min, 0.5 ,uMO,/min respectively. The data obtained for the motility in each of the inhibition steps are inserted in Fig. 5. Both the average velocity, B, and the number of sperm, c, which are moving, are shown decreasing with the higher amytal concentration. Even though the photoelectric method used for measuring motility, does not allow quantitative determination of the halfwidth of the velocity distribution [14], it still indicates qualitatively the shape of this distribution. It was observed that at each of the “inhibition steps”, a “bell shaped” velocity Experimental
Cell Research
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Respiration
and motility
of spermatozoa
319
distribution is present. The consequences of this observation are discussed on p. 323. The results of 10 different experiments were consistent enough to allow them to be pooled. Fig. 6 shows the effect of amytal on the respiration of the 10 samples. The respiration is plotted as the fraction of the value for the
0
2
4
6
8
IOmM
Fig. 8.-Change in average velocity in ten different sperm samples, as a function of the amytal concentration. Amytal
concentration
uninhibited preparation. Fifty per cent inhibition is obtained at an amytal concentration of approximately 1.2 mM. Fig. 7 represents the effect of amytal on the number of moving sperm. The same correction procedure as described on pp. 316-317 (Fig. 4) was applied here. It can be seen that when 90 per cent inhibition of the respiration is obtained, at around 10 mM amytal, only about 25 per cent of the number of original cells are still moving. This curious effect is referred to in more detail in the discussion. Fig. 8 shows the decrease of the average velocity with increasing amytal concentration. As the initial velocity does not show a big spread for the different samples the data are plotted absolutely. In Fig. 9 all data points have been pooled together and plotted as respiration in pMO,/min versus number of moving cells X average velocity. All data have been presented here by their absolute value, without any of the previously described correction applied. Fig. 9 shows that in the sperm system used the “amount” of motility, expressed as (cu) is in direct relation to the respiration. The relation holds over a range of respiration values of 10 ,&lO,/min to 0.3 pMO,/min, and of “motility” values from 240 to 6 X 10’ Experimental
Cell Research
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320
R. Rikmenspoel
sperm ,u/sec. This represents roughly a range of a factor of 30. This can be considered as evidence that the amytal has no “poisoning” effect but only restricts itself to inhibition of the respiratory chain with a consequent reduction of production of free energy available to the sperm. The straight line drawn in Fig. 9 which best fits the measured points leads to a relation where
Fig. 9.-Respiration of ten different sperm samples plotted versus the “amount” of motility, expressed as number of moving sperm x average velocity. The variation within the samples is obtained by stepwise inhibition by amytal (cf. Fig. 5).
c / o,,LLLLI_LLL_I
Lllll’
I I III”!
IO
Concentrolion
102
in lO’/ml
x average
I03
velocity
in p/set.
respiration is proportional to (c X u)p, p being 0.95. In view of the large scattering, the data of Fig. 9 cannot be considered inconsistent with p = 1. If the respiration R is expressed in mol O,/sec, c in number of sperm, and v in cm/set, the relation can be written as R = a(c x v)molO,/sec
(1)
CChaving the value of a = 6.1 X lo-16 mol Oz/sec/sperm/cm/sec. Kinetics of amytal action.-Determination of the speed with which amytal takes effect is important because it gives information about the permeability of the membrane of the sperm. By the delay between the decrease in respiration and that in the motility, it sets a limit to the amount of energy rich bonds (,P) which are stored in the sperm. The measuring chamber described is not suitable for measurement of fast changes in respiration. When additions are made in the cuvet, or when the slide is flushed, violent irregular signals occur which overload the amplifier used. Therefore, for measuring the speed of change of respiration Experimental
Cell Research
37
Respiration
and motility
321
of spermatozoa
upon addition of amytal, the oxygen electrode was operated in a separate cylindrical glass cuvet, as described by Chappell [5]. It was found that in these conditions the indication time of the electrode was the limiting factor in determining the speed of action of amytal. This is illustrated in Fig. 10, which shows that a new steady state of respiration is indicated by the electrode
I
Amytal IO mM
1
‘~‘402
*
Fig. lO.-Tracing of the oxygen electrode during the addition of amytal. The sensitivity and the time-scale are greatly expanded compared to those in Figs. 2 and 5. The jump in oxygen level represents the amount of oxygen dissolved in the amytal solution.
: I
; ’
it
60 sec.
*
within 10 set after the addition. In 4 experiments performed the results were identical; the new steady state is reached within the indication time of the oxygen electrode. In order to obtain information about the permeability of the membrane, an indication time of at least 10 x faster than now available would be required. The kinetics of the motility change upon amytal inhibition was studied using the micro chamber and the photographic “track” technique. The I. Delay z between the addition of amytal and the moment readjustment to the inhibited steady state has occurred.
TABLE
Amytal addition m&f
7 initial Wet)
v final Wsec)
t (set)
1 2 3
1.5
78 74 80
26 27 24
88 70 46
4 5 6
10
92 98 80
55 52 45
45 53 70
Experiment No.
Experimental
that 90 %
Average of t (set)
68
56 1 Cell Research
37
322
R. Rikmenspoel
procedure was as follows: after the sample in the chamber was warmed up to the point where constant motility was reached, the camera was put in place and focussed. The addition of amytal was made with one of the Hamilton syringes. Three seconds were allowed for stirring of the content of the cuvet, and then the slide was flushed by means of the second syringe.
Amytal 110
t
Seconds
Fig. Fig. Il.-Change amytal.
with
time
Fig.
11. of the average
velocity
of a sperm
sample
upon
12. addition
of 1.5 mM
Fig. 12.-Schematic form of the function g(u). The peak at v = o represents the cells which have become motionless upon addition of amytal. The cross-hatched area represents the fraction of the cells, having an original velocity u,,, which is still moving after the amytal addition. The dotted peak at u,, shows the cells before the amytal addition.
Photographs of the slide were made at 5 set interval, starting 10 set after the amytal addition. One minute after the addition the interval between photographs was increased to 10 sec. At each photograph 6 to 14 tracks were recorded from moving sperm. This is sufficient to define the average velocity u of the sperm at that moment to about +lO p/set. It was not considered that figures about the average number of moving sperm can be deduced from so few recorded tracks, so only the decrease of u with time is reported. Two groups of 3 experiments each were performed. The first group was aimed at approximately half inhibition by the addition of 1.5 mM, and the Experimental
Cell Research
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Respiration
and motility
of spermatozoa
323
second group at maximal inhibition by addition of 10 mM amytal. As the contents of the cuvet were probably not completely mixed during the 3 set allowed for stirring, the actually measured inhibition of v should not be read as being quantitatively related to the amytal concentration used. Fig. 11 shows the results of a partial-inhibition experiment. Table I summarizes the results of the 6 experiments performed. It can be seen that the new, inhibited, steady state is reached approximately 1 min after the addition. There is no apparent difference between the two groups.
DISCUSSION
The action of amytal.-The data presented in Figs. 6, 7 and 8 show that the action of amytal on sperm motility is a complicated one. As mentioned before, it cannot be assumed that the addition of a certain concentration of amytal changes the original velocity v, of a sperm into v = tlvO, tc being the same for all sperm. In that case no decrease in the number of moving cells c should be found, as the velocity distribution simply would shift towards the ordinate. In general we can say that a sperm having originally a velocity v, will have a chance g(v) of moving with a velocity v after the addition of amytal. Qualitatively one can say about the function g(v): 1. For v = 0, which represents sperm that have become motionless, g(v) has a value +O. So Jg(v)d v, excluding the region near v = 0, should have a value < 1. 2. As the velocity distribution after the addition of amytal still has a “bell shape”, with small values for the lower velocities, it follows that the function g(v) has to be small for the lower velocities also. Obviously g(v) also has a “bell shape”, qualitatively sketched in Fig. 12. The three important quantities of g(v) are: the fraction A of cells which still move, represented by the cross-hatched area in Fig. 12, the mean, B, of g(v) and its half width y. A, B and y are of course functions of the original velocity v,. If the form of the velocity distribution of the sperm before and after addition of amytal is known, it is possible to solve A, B, and y in first order dependence on v,. Using the photographic method, experiments to measure the form of the velocity distributions are presently being undertaken, with the aim of obtaining quantitative information on A, B and y. Energy considerations.-The data presented in Fig. 9 and on p. 320 can be regarded as strong evidence that the motility is directly dependent on the steady state level of ATP production. If the coupling of the oxidative Experimenlal
Cell
Research
37
R. Rikmenspoel
324
phosphorylation were maximal, at a P/O ratio = 3, the maximal amount of free energy, F, available to the sperm in our preparation can be calculated from eq (l), p. 320, assuming 9 kcal to be liberated at hydrolysis of 1 mol ATP [S]: F = 1.4 X 10-S (c x u) erg/set. (2) with c being the number of moving cells, and u the average velocity in cm/set. Taylor [24] has estimated the amount of work performed hydrodynamically by a moving flagella. Using as a model for the flagella a thin rod in which flat sinusoidal, flexural, waves are traveling, Taylor finds W = 4n3,uf2b2L/ (0.62-ln(ne/l) where: p = viscosity of the fluid; f = frequency of the tail wave; b = amplitude of the tail wave; 1 = wavelength of the tail wave; L = length of the tail; Q = diameter of the tail. The sperm observed in our experiments, however, show a three-dimensional tail wave. It has been shown recently [17] that the two components of the helical wave have amplitudes which have a ratio of 2 to 1. In the first order approximation used by Taylor the work performed by the two components of the helical wave can be taken to be linearly additive [24]. This yields for the hydrodynamic dissipation of the sperm studied in this paper: W=-
6 n3/xf2bzL 0.62 - ln(ne/il)
Carlson [3], using a more complicated model for the flagella, arrived at essentially the same results as Taylor. The following values have been adopted for some of the quantities appearing in eq (3): p = 1.0 X 1O-2 poise [16]; L = 60 ,u; 3, = 35 ,u [14]; Q = 0.5 ,L For rotating cells u has been found previously [ 121 to be: v=eb2, being E = 1.2 X 104/cm/set. Data on fare not as well defined. For non-rotating bull sperm it is reported [14] that f * 22 cps, with no clear relation between f and v. Gray has reported lower values [7] for f, f = 10 cps in non-rotating bull sperm. It has been shown recently, however, that this is due to insufficient time resolution of the cinemicrographs from which these data were obtained. From cinemicrographs with a time resolution of 5 msec it was found by the author that for 10 rotating cells the frequency of the tailwave was between 20 and 24 cps [17]. For these 10 cells no correlation between f and v was found. As an average E
Experimental
Cell Research
37
Respiration
and motility
of spermalozocr
325
figure is used here: f = 22 cps. It should be realized, however, that some uncertainty exists as to the correctness of the mean value for /as used here. If all these values are inserted in eq (3) we find that the work \\’ performed by a number c of sperm is \V= 2.25 X 1O-4 (c X II) erg/set. The energy dissipated in the elastic deformation of the tail has to be taken into account. Jlachin [lo] has calculated that the amount of work needed for elastic deformation of the tail is :f- of the hydrodynamic dissipation. This brings the total work \V tOt performed by moving sperm to
nyt,,= 3.0 x lO-4
(cx 0) crg/sec.
(4)
A comparison of equation (2) and (4) indicates that the “efficiency” of the oxidative metabolism is at least about 22 per cent. This figure is arrived at by assuming the oxidative phosphorylation to be tightly coupled. Chance [4] has pointed out, however, that under conditions of less tight coupling the thermodynamic efficiency may be lowered, but the amount of ATP produced for a ccl1 may actually be higher, due to the much faster turnover. In view of the wide range over which the apparent efficiency reported here seems to be constant, one would assume that under our conditions the coupling has remained fairly constant. Actually measured P/O ratio’s in sperm should, therefore, be of great interest. Energy reseraes in sperm.-The results of the esperiments on the kinetics of amytal action as illustrated in Fig. 10 show that the electron flo\v in the respiratory chain is readjusted within 10 set after the addition of amytal. The production of ATI’ is readjusted at Lhe same time to its new level. The slojver decay of the motility (Fig. 11) can be seen as an exhausting of reserves of ATI’ available in the sperm. From the data of Fig. 11 and Table I it can be very roughly concluded that stored energy in our sperm preparation is sufficient to maintain motility over a period of the order of 30 sec. According to cq (4) the amount of work performed by one normally moving sperm in that period \\ould be w 10-O erg. Hornstein and Steberl ;2: have reported \:alues of =\‘l’P content of bull semen of approximately lo-13 g ATP/sperm. The free energy obtainable by hydrolysis of this amount would come to 0.8 x 1V4 erg/sperm. A comparison of this figure \vith the amount of \vork performed as mentioned above (w 1W4 erg/sperm) is again a confirmation that the motility of sperm is directly dctcrminrd by the instantaneous amount of ATP made available to the sperm. Experimenld
Ceil
Research
37
R. Rikmenspoel
326
SUMMARY
Simultaneous measurements of motility and respiration of spermatozoa have been performed using a specially developed microchamber. Of each sperm sample used the respiration and the motility was stepwise reduced in the course of the experiment by additions of amytal. Kinetic studies showed that after the addition of amytal the new steady state level of respiration is reached within 10 set and that of the motility in about 60 sec. Steady state values of respiration plotted versus the steady state values of number of moving sperm X average velocity show an almost linear relation, which holds over a range of a factor 30. From this relation can be deduced that the amount of energy needed for the sperm motility is at least 22 per cent of the free energy which can be produced by the oxidative metabolism. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
10. 11. 12. 13.
14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
BISHOP, D. W., Physiol. Rev. 42, 1 (1962). BOMSTEIN, R. A. and STEBERL, E. A., Expfl Cell Res. 18, 217 (1959). CARLSON, F. D., Proc. 1st. Nat. Biophys. Conf. Yale Un. Press, 1959. CHANCE, B. and WILLIAMS, G. R.. Adu. Enzumol. 17. 65 (1956). CHAP&L, B., in T. W. Gobnwr~‘and O.Lr~“o~mm (ids.): Biological Structure and Function. Academic Press, New York, 1961. GONSE, P., in D. W. BISHOI~ (ed.), Spermatozoan Motility. AAAS, Washington, D.C., 1962. GRAY, J., J. Exptl Biol. 35, 96 (1958). LEHNINGER, A. L., Rev. Mod. Phys. 31, 136 (1959). LINDAHL, P. E. and BRATTSAND, R.. Zool. Bidraa. Uam-ala 35. 505 11962). MACHIN,‘K. E., J. Exptl Biof. 35, 796 (1958). ” ” NELSON, N., J. Biol. Chem. 153, 375 (1944). RIKMENSPOEL, R., in D. W. BISHOP (ed.), Spermatozoan Motility. AAAS, Washington, D.C., 1962. __ Reu. Sci. Instr. 35, 49 (1964). __ ibid. 35, 52 (1964). ~ Experienfia 13, 124 (1957). __ Photoelectric and Cinematographic Observations of the Motility of Bull Sperm Cells. Thesis, J. A. Smit, Utrecht, 1957. ~ Trans. N.Y. Acad. Sci., Series II 26, no. 8 Suppl. (1964). In press. RIKMENSPOEL, R., VAN HERPEN, G. and EIJKHOUT, P., Phys. in Med. and Biol. 5,167 (1960). ROTHSCHILD, LORD, Proc. Roy. Sot. B 151, 1 (1959). __ in G. E. W. WOLSTENHOLME, M. P. CAMERON and J. S. FREEMAN (eds.), Mammalian Germ Cells. J. &. A. Churchill, London, 1953. ROTHSCHILD, LORD and CLELAND, K. W., J. Ezpfl Biol. 29, 66 (1952). SHEPARD, C. S. and MOTTLE, G. A., .I. Biol. Chem. 179, 369 (1949). SOMOGYI, M., J. Biol. Chem. 160, 61 (1945). TAYLOR, G. I., Proc. Roy. Sot. A 211, 225 (1952).
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