The control of bull spermatozoon flagellar activity has been investigated using direct current injection into the cells through an impaling glass microelectrode. Negative current injection results in a decrease in the flagellar frequency. Flagellar frequencies can be decreased to zero with high negative currents. This current injection response is dependent on the magnesium concentration available to the spermatozoon interior. The current injection response is nearly independent of ATP concentrations. Resistance measurements indicate that the current injection pathway has a resistance of about 200 ± 300 kΩ, and that the current flowing through the cell membrane is not exceedingly large. Measurements of the induced potentials indicate transmembrane potentials during current injection of about —35 ± 30 mV per μA of injected current. The results are compatible with an active transport process in bull spermatozoa that controls the flagellar activity in response to current injection by decreasing the internal Mg2+ concentrations during the injection of current.

Spermatozoa do not appear to have a mechanism for control of the level of flagellar activity. The motility of intact spermatozoa is constant over a considerable period of time (Gray, 195 5; Rikmenspoel, Sinton & Janick, 1969; Mann, 1964). The variation in activity of cellular flagella, which points to a control mechanism within the cell body, is not observed in spermatozoa.

Demembranated sea-urchin as well as bull spermatozoa show a constant flagellar activity that appears approximately normal upon the addition of external ATP and Mg2+ (Gibbons & Gibbons, 1972; Lindemann & Gibbons, 1975). Bull spermatozoa which have been impaled with a glass microelectrode display coordinated flagellar wave motion upon the addition of external ATP and Mg2+ (Rikmenspoel, Orris & O’Day, 1978). The intensity of the flagellar motion in sea-urchin or bull spermatozoa does not appear to be influenced by Ca2+ (Gibbons & Fronk, 1972; Rikmenspoel et al. 1978). It has recently been found by Brokaw & Simonick (1977), however, that the waveshape of demembranated sea-urchin sperm flagella can be affected by Ca2+.

During the course of experiments in this laboratory it was noticed that bull spermatozoa often show a perceptible change in flagellar motion within 1 s of impalement by a microelectrode. This change occurs within a time much shorter than would be necessary for a change in motion due to a change in concentration of chemical substances inside the sperm by diffusional exchange with the outside medium. The characteristic time of this latter process is of the order of 30 s (Lindemann & Rikmenspoel, 1971; Nevo & Rikmenspoel, 1970). A change in the internal electrical resting potential of a spermatozoon upon impalement can easily take place in time intervals of much less than 1 s (Larsen, Nielsen, Pape & Simonson, 1971). The immediate change in sperm flagellar motion upon impalement therefore suggests that the internal potential can influence the flagellar motion.

In this paper experiments are described in which bull sperm flagellar motion was controlled by electrical direct current injected through an impaling microelectrode. The results indicate that the control was mediated by means of the Mg2+ ion. Preliminary communications of part of the results in this paper have been made (O’Day, Rikmenspoel & Lindemann, 1976; O’Day & Rikmenspoel, 1977).

Sperm preparations

Bull spermatozoa were generously provided by the Eastern Artificial Insemination Cooperative at Ithaca, New York. After ejaculation the semen was diluted 5 times in an optically clear 8gg yolk/citrate diluent (Rikmenspoel, 1957), cooled to 4 °C and transported to the laboratory.

The spermatozoa were washed twice by centrifuging and resuspending them in a basic suspension medium (BSM) described below. For experiments, one or two drops of the washed sperm suspension were added to 2 ml of a basic experimental medium (BEM) described below. This highly dilute suspension in BEM was placed in a microscopic slide chamber described previously (Lindemann & Rikmenspoel, 1971).

Experiments were generally performed at a room temperature of 21 ± 1 °C except in the temperature-dependence studies. Temperatures in the preparations were measured with a Telethermometer Model 42SL (Yellow Springs Instrument Co., Yellow Springs, Ohio).

Preparations of spermatozoa at room temperature retained approximately constant flagellar activity (estimated visually) for as long as 20 min, which allowed for the investigation of 4 to 5 cells per preparation. After 30 min, the flagellar frequencies of intact cells were often much reduced, and the percentage of cells that were motile was also reduced. Preparations were generally changed after 10 to 20 min.

Media and chemicals

The basic suspension medium (BSM) used for washing the sperm consisted of 163 mM sucrose, 72 mM KC1, 7 · 2 mM Na-lactate, 2 mM Na phosphate buffer, 6 μM CaCl2 and 4·2 mM MgSO4. The pH was adjusted to 7·3. When the experiments called for low Mg2+ or Mg’+-free conditions, the MgSO4 was omitted from the BSM.

The basic experimental medium (BEM) used to suspend the sperm during experiments consisted of 169 mM sucrose, 75 mM KC1 and 3 mM Na phosphate buffer, at pH 7 ·3. Additions to the BEM, as required by the experiments, were made by Hamilton syringe.

ADP (disodium salt) was obtained from Sigma Chemical Co. (St Louis, Missouri); ATP (disodium salt) from Boehringer Mannheim Co. (New York, N.Y.). Solutions of 0 ·1 M ADP and of 0 ·1 M ATP at pH 6 · 8 were frozen and stored at —20 °C in aliquots of 1 ml. Additions of ADP or ATP to the BEM were made from aliquots thawed just prioi to use. Mg2+ was added from 0 ·1 M solutions of the sulphate salt.

Dithiothreitol (DTT) was obtained from Sigma Chemical Co. (St Louis, Missouri). The DTT was added to the BEM, when required, from frozen aliquots of 0 ·1 M concentration at pH 6 · 8, which had been thawed just prior to use.

Apparatus and procedures

Microelectrodes

Glass microelectrodes were made from No. 9530 glass capillary tubing (Coming Glassworks, Coming, N.Y.) with a Chowdhury pipet puller (Chowdhury’, 1969). The microelectrodes were filled by vacuum with methanol, and transferred to a solution of 2 M KC1. The tip diameters were measured to be 0 · 15 ±0 03 μm (mean and S.D.) with a scanning electron microscope. The electrode resistances were typically of the order of 10 to 20 MΩ.

Current injection

Electrical current was injected through the impaling electrode from a variable d.c. source with an internal resistance of too MΩ.

Resistance measurements

Resistance measurements were made by means of an a.c. Wheatstone bridge. The 2 fixed bridge arms had a resistance of 20 MΩ. The variable arm was a parallel combination of a resistance and a capacitance. The resistance could be varied from o to 120 MΩ and the capacitance could be varied from 20 to 400 pF. The variable capacitance was needed to balance the stray capacitance of the microelectrode + spermatozoon. By trial and error optimal functioning of the bridge was found with a sinusoidal input of approximately 9 Hz. When the total resistance in the unknown (electrode) arm was 10 to 20 MΩ, changes in resistance could be measured to an accuracy of approximately 30 kΩ.

Impalement procedures

The methods used for impaling bull sperm heads have been described in detail previously (Lindemann & Rikmenspoel, 1971).

Potential measurements

Measurements of the potentials induced in the preparation by the current injection microelectrode were made using a second KCl-filled glass microelectrode mounted so that the 2 microelectrodes were facing one another under the microscope objective (Fig. 1). The potential measuring electrode was controlled by a Brinkman CPVI micromanipulator and a piezo-electric advancing apparatus. Dual impalement of a spermatozoon was a difficult procedure, and the majority of attempts were unsuccessful. However, with practice it was possible to make dual impalements, somewhat regularly, at a desired microelectrode separation. The second microelectrode was connected via a Ag-AgCl wire to a Model M4 amplifier of W-P Instruments Co. (Hamden, Connecticut). This amplifier measured the potential difference between the microelectrode tip and the reference electrode. The bathing solution had to be grounded carefully, and the Ag-AgCl reference electrode had to be replaced often due to the loss of the chloride coating by electrolysis. If the bath was not well grounded, the measured potentials would show slow response to injected currents, presumably due to the simultaneous charging of the bathing solution. The injected current and the resulting potentials were recorded using a dual trace strip chart recorder.

Motility measurements

The motile activity of the spermatozoa was expressed in terms of the frequency of the flagellar beat. At the room temperature at which the experiments were performed this frequency was maximally around 6 Hz. The beat frequencies were obtained by timing 10 beat cycles with a stopwatch. With some practice it proved possible to measure reliably frequencies up to slightly more than 5 Hz. From repeating the measurement on the same spermatozoon we have estimated that the accuracy of this frequency measurement was around ±0 · 3 Hz. Frequencies above 5 · 5 Hz carry an error which is probably considerably higher. Frequencies of <0 · 2 Hz were rounded off to zero.

Cinemicrography

Films were taken when desired at 20 or 50 frames/s with a Millikan DBM5-C camera (Teledyne Corp., Arcadia, CA) on Kodak Plus-X film. The procedure for frame-by-frame analysis has been described previously (Rikmenspoel, 1965).

During the early stages of the experiments external ADP was used as a power source for maintaining the flagellar activity of the spermatozoa after impalement with a microelectrode. It had been found (Lindemann & Rikmenspoel, 1971) that externally added ATP inhibited the flagellar activity after impalement. Later, with presumably more pure ATP from a new’ source, post-impalement flagellar activity could be maintained by ATP (Rikmenspoel et al. 1978). Most subsequent experiments were done with ATP as external power source.

Effects of current injection

Bull sperm in BEM with 4 mM ADP, 0 · 5 mM Mg2+ and 1 mM DTT reached a steady state flagellar frequency of approximately 2 · 7 ± 0 · 9 Hz in 30 to 60 s after impalement. When negative direct current (cathode inside the cell) was injected through the impaling electrode the flagellar frequency of the sperm decreased to a new steady state level that was dependent on the amount of current being injected. The time required for the flagellar frequency of the sperm to reach the new steady state was about 20-40 s. However, these times were often shorter when sufficient current was injected to cause the flagellum to cease beating completely. On termination of the injection of negative current the flagellar frequencies returned to values near the original postimpalement values. A decrease in flagellar frequency during negative current injection followed by a near complete recovery upon termination of current injection could be observed several times in succession in the same sperm. In some cases as many as ten of these cycles have been observed. The effect was thus quite reversible.

The amplitude of the flagellar wave was frequently, but not always, reduced during the injection of negative current. Upon termination of the current injection the amplitude often did not recover its original value. Since this effect was difficult to quantitate, and because it was not well reproducible, only the flagellar frequency was used to express the flagellar activity below.

Fig. 2 shows the flagellar frequency as a function of the amount of injected negative current for bull sperm in BEM with 4 mM ADP, 0 · 5 mM Mg2+ and 1 mM DTT. The frequencies are expressed as a fraction of the original post-impalement frequency for each case, to reduce the scatter. Inserted in Fig. 2 are the results of experiments in which the microelectrode was removed from the sperm head after impalement and placed at a distance, d, of 1 μm from the spot of impalement outside the sperm head (×). Fig. 2 also shows the results of experiments in which negative current was applied with the tip of the microelectrode at a distance d = 1 μm from the edge of an intact (not impaled) sperm head (®). Fig. 2 show’s clearly that only negative current applied to the inside of a sperm has the effect of decreasing the flagellar frequencies.

The negative currents applied in the above experiments appear to be very large for an organism as small as a bull sperm. However, in the section Resistance measurements below it is shown that a very low resistance path exists between the tip of the impaling microelectrode and the external fluid bath. Almost all of the injected current passes through this path, which presumably represents the hole made by the impalement and through which a free diffusional exchange of the various substances can take place (compare Lindemann & Rikmenspoel, 1971). In the section Potential measurements below, it is shown that the potentials induced by the injected currents are of the order of 50 mV. These measurements, taken together with the reversible nature of the current injection response, indicate that the current injection response does not involve exceeding the current-carrying capacity or the electrical-breakdown limit of the membrane of the sperm, and that the magnitude of the currents used should not be considered alarming.

The injection of positive direct current in impaled bull sperm produced effects different from those produced by the injection of negative current. The flagellum appeared to stiffen upon injection of positive current and a very sharp kink developed in the flagellum about three quarters of the way down the midpiece toward the tail-midpiece junction. This kinking was often accompanied by a curling of the very tip of the flagellum. This response to positive current injection did not lend itself to quantitation and it was difficult to interpret. It was therefore not pursued beyond these qualitative observations.

In the subsequent sections below the response of flagellar activity to the injection of negative direct current is further investigated and quantitated.

Mg2+ dependence

Fig. 3 shows the flagellar frequency as a function of the injected negative current for bull sperm in BEM with 4 mM ATP, 1 mM DTT and 3 different Mg2+ concentrations. The Mg2+ ions can bind to the ATP present in the medium. In Fig. 3 and subsequently in this paper the Mg2+ concentration always refers to the concentration of Mg2+ added to the medium and not to the concentration of free Mg2+. In the Discussion the binding of Mg2+ to ATP will be referred to in more detail.

Each of the 3 curves in Fig. 3 has roughly the same monotonic shape. The current, , that reduces the flagellar frequency to half the original post-impalement frequency was chosen as a characteristic for the curves.

In Fig. 4A are shown the values for as a function of the external Mg2+ concentration for spermatozoa powered by 4 mM ADP and by 4 mM ATP. It can be seen in Fig. 4A that , increases smoothly with the Mg2+ concentration. No systematic difference in for the ADP and the ATP powered sperm is apparent in Fig. 4A. The values for obtained at an external ATP concentration of 0 · 5 mM are also inserted in the figure. No clear indication of dependence of on the ATP concentration is shown in these results.

In Fig. 4B are shown the post-impalement frequencies, in absolute value, of the sperm powered by 4 mM ADP and by 4 mM ATP as a function of the Mg2+ concentration. The sharp rise of the flagellar frequency at low Mg2+ concentration (< 0 ·1 mM), followed by a rather flat plateau has been reported previously (Rikmenspoel et al. 1978).

The data in Figs. 3 and 4 A suggest that the Mg2+ ion is involved in mediating the current injection response of the spermatozoa. However, the fact that the postimpalement flagellar frequency as shown in Fig. 4B is rather independent of the Mg2+ concentration for concentrations above 0 ·1 mM and the fact that no threshold behaviour of the frequency as a function of injected current is apparent in Fig. 3 indicate that an interpretation of the role of Mg2+ should be made cautiously. We shall come back to this question in the Discussion.

ATP dependence

Fig. 5 A shows the dependency of (measured as described above in the section Mg2+ dependence) on the concentration of external ATP for impaled bull sperm in BEM with 0 ·5 mM Mg2+ and 1 mM DTT. Even though the data in Fig. 5 A suggest a slight decrease of with the ATP concentration, can be regarded as largely independent of the external ATP concentration.

Fig. 3B shows the post-impalement flagellar frequency of the sperm as a function of [ATP]. The shape of the curve in Fig. 3B is very similar to that of Fig. 4B above, which shows the Mg2+ dependence of the flagellar frequency: a rather steep rise at low concentrations followed by a gradually declining plateau.

Fig. 4A above showed that the manner in which depends on the Mg2+ concentration is essentially the same for ATP concentrations of 0 · 5 mM and 4 mM. Together with the data of Fig. 5 A this indicates that the ATP is not involved in the mechanism for the response of the sperm flagella to negative current injection either directly or through an interaction with Mg2+.

Resistance measurements

All resistance measurements were made with the a.c. Wheatstone bridge on spermatozoa in BEM with 4 mM ATP, 0 ·5 mM Mg2+ and 1 mM DTT. In the course of the experiments it was noticed that a large change in the apparent microelectrode resistance occurred when the microelectrode tip touched the microscope slide. In all resistance measurements reported below, the stage of the microscope holding the sample was therefore lowered by a few microns after the impalement of the sperm. The impaled sperm usually stuck to the microelectrode, and neither the sperm nor the microelectrode was then in contact with the slide.

The change in resistance upon impalement was measured as + 2ro ±310 KΩ (mean and S.D. over 24 cells). In each case the impaled spermatozoon was removed after the resistance measurement from the impaling electrode by gently tapping the micromanipulator. The resistance change upon removal was measured as —140+ 190 KΩ (mean and S.D. over 24 cells).

The resistance changes found in the present experiments are much smaller than the values reported earlier from this laboratory (Lindemann & Rikmenspoel, 1971, 1972) which were of the order of 20 MΩ. We presume that this difference is due to the fact that in the earlier experiments no attention was paid to whether the electrode tip touched the slide during the impalement. Furthermore, in these earlier experiments electrodes of a larger tip diameter were used (0 · 3–0 · 5 μm) which made it more likely that the glass slides were touched during impalement of the sperm heads.

Our measurements indicate that the resistance from the tip of the impaling electrode to the external bath is of the order of 200 KΩ. This very low resistance represents not a membrane resistance of the impaled spermatozoon but most probably a short circuit path through the leaky junction between the microelectrode and the sperm membrane. That this junction does not seal is conclusively shown by the free exchange of ATP between the inside of the sperm and the external medium.

The specific resistance of the bull sperm membrane is not known. In other organisms values of the membrane resistance varying from 103 Ω cm2 for the Loligo axon (Cole & Curtis, 1939) to 2 × 106 Ω cm2 for the Chara plasma membrane (Gaffey & Mullins, 1958) have been reported. If we take a lower limit for the specific resistance of the bull sperm membrane of 103 Ω cm2, the total membrane resistance is 7 × 108 Ω (with a spermatozoon surface area of 140 μm2). Therefore the fraction of the injected current in our experiments that flows through the membrane of the sperm is less than 3 × 10−4, and the remainder of the current passes through the short circuit path around the microelectrode.

Potential measurements

The electrical potentials induced near the tip of the microelectrode used for current injection were measured with a second microelectrode as described in the section Experimental methods. The distance between the tips of the 2 microelectrodes was measured with an eyepiece micrometer. In all measurements the bath fluid was BEM with 4 mM ATP, 0 ·5 mM Mg2+ and 1 mM DTT.

The bath fluid used has a lower ion concentration than either Ringer solution or typical suspension media for marine organisms. Consequently, it cannot be assumed that its conductivity is high and that no potentials are induced in the neighbourhood of the tip of the current electrode. A measurement of the voltages induced in the fluid bath (with no sperm present) when current was applied through the current electrode, showed that the tip of the current electrode acted as an electrical monopole, and that significant potentials were induced to distances of approximately 20 μm.

Thus, potentials induced by injected current in a spermatozoon have to be corrected for the potentials induced in the external bath. The procedure used in potential measurements was as follows. A sperm was impaled by the current injection microelectrode in the acrosomal region of the head. The tip of the potential measuring electrode (PE) was then positioned at a chosen distance from the tip of the current microelectrode near the neck region of the head. Three different amounts of current were injected, and the induced potentials were recorded. The sperm was then impaled with the electrode PE. Care was taken that the electrode PE was advanced in such a way that the separation of the tips of the 2 microelectrodes remained approximately the same. With some practice this proved to be quite possible. The induced potentials were then recorded for 3 values of injected current. The electrode PE was subsequently withdrawn, and placed in the original position. In this situation the induced potentials were again measured for 3 values of injected current.

In all cases the potential measured was found to be proportional to the injected current. The results of the experiment can therefore be characterized by the value of the potential induced per μA of injected current. The induced potential per μA of injected current after the electrode PE had been withdrawn from the sperm were found not to be significantly different from those before the electrode PE had impaled the sperm. Apparently the hole in the sperm head made by electrode PE does not appreciably affect the potential field outside the spermatozoon.

Fig. 6 shows the induced potential per μA of injected current with the voltage electrode PE outside the sperm and with PE inside the sperm. It can be seen in Fig. 6 that the potentials inside the sperm were consistently higher than those outside for the same electrode separation. Extrapolation of the lines in Fig. 6 to separations larger than 9 μm suggests that the potential difference between the inside and outside of the sperm persists well beyond the head-midpiece junction.

The data shown in Fig. 6 cannot be interpreted as a precise measurement of the transmembrane potentials induced in a sperm. The data can be taken, however, as a firm indication that potential differences between the inside and the outside of a sperm are induced by current injection. Based on these results we estimate the order of magnitude of the induced potential differences from Fig. 6 to be about —35 ± 30 mV per μA of injected current. This is well within the physiologically meaningful range.

The current injection response was unchanged by the presence of the second impaling microelectrode. The value of for cells impaled with the second micro-electrode was —0· 48 ± 0· 26μA at 0· 5 mM external Mg2+ (17 cells; errors estimated as for Fig. 4); that for cells impaled only with the current injection microelectrode was —0· 51 ± 0· 07μA.

Temperature dependence

The temperature dependence of the time course of the response of the flagellar activity to negative current injection was investigated for sperm in BEM with 4 mM ATP, 0· 5 mM Mg2+ and 1 mM DTT. At temperatures above room temperature the sperm preparations tended to decay rather quickly. Accordingly the preparations were renewed frequently.

In all cases the injected current used was —i·8 μA, to ensure that the flagella came to a complete stop. The injection of current was terminated a few seconds after the flagella had become motionless. Films were taken continuously at 20 frames/s (for T = 19 °C) or at 50 frames/s (for T = 25· 5 and 37 °C) from a few seconds before the start of current injection to the time the sperm had recovered steady activity after the termination of current injection (estimated visually).

Fig. 7 shows the frequency as a function of time during the course of typical experiments at 25· 5 and at 37 °C. It can be seen in Fig. 7 that the post-impalement frequency (before the start of current injection) increases with temperature. At all temperatures the flagellar frequency decreased at first rather gradually after the start of the current injection, and then fell abruptly to zero. This indicates a threshold behaviour. The time from the start of the current injection to the complete halt of flagellar activity has been taken as the characteristic time, ts, for the process.

When the current injection was terminated the flagella remained at rest for a period of time and then rapidly increased activity until the steady state recovery level of flagellar frequency was obtained, as illustrated in Fig. 7. The recovery from the effects of current injection thus also shows a clear threshold behaviour.

Table 1 lists the average post-impalement frequency before the start of the current injection and the average of ts at the three temperatures of 19, 25 · 5, and 37 °C. The data in Table 1 indicate that the flagellar frequency has a Q10 of 1·5 ± 0·5, whereas the Q10 for the velocity of the flagellar stoppage during current injection is 2·6 ± 0·5. The dissimilarity of these two Q10 values suggests that the current injection response operates by a mechanism different from that of the flagellar contractility.

The results of the experiments described above have established that it is possible to control the flagellar activity of bull sperm by means of injecting negative current through an impaling electrode. This does not by itself show, however, that the experiments simulate a ‘natural’ mechanism which has physiological importance. In the following discussion we will consider what conclusions can be made about the mechanism through which the electrical control of the flagellar activity operates.

The magnitude of the currents used in the experiments was very large indeed (of the order of 1 μA) for an organism as small as a spermatozoon. By comparison, the currents employed in the electrical control of ciliary motion in Paramecium (see the review by Naitoh & Eckert, 1974) were in the order of several nA. The surface area of a Paramecium is typically 104μm2. Since in Paramecium the plasma membrane seals around the impaling electrode (Eckert, 1972) all of the current must pass through the membrane. The resultant current density at the Paramecium membrane is thus several × 10−6 A/cm2.

It appears well established now that in very small cells the membrane does not seal around an impaling microelectrode. This has been observed in Euglena (Nichols & Rikmenspoel, 1977), in Ehrlich ascites tumour cells (Larsen et al. 1971), as well as in bull sperm (Lindemann & Rikmenspoel, 1971). The resistance measurements described above, confirm that in the present experiments practically all of the injected current is short circuited through the leakage path around the impaling microelectrode. The estimated upper limit for the current density in sperm membranes in the present experiments, made in the section ‘Resistance measurements’, was 2 × 10−4 A/cm2. This is not much larger than the current density in the experiments on Paramecium discussed above.

The observations that the effects of current injection in the present experiments were fully reversible, and that they could be repeated several times on the same spermatozoon, support the conclusion that the currents used did not cause damage to the sperm.

The Mg2+ ion is clearly involved in the mechanism of the current injection response. The data in Figs. 3 and 4 suggest that the effect of current injection is to reduce the internal Mg2+ content of the impaled sperm, which in turn reduced the flagellar frequency. The results in Fig. 4B, which are in agreement with earlier results in this laboratory (Rikmenspoel et al. 1978), imply that the internal Mg2+ concentration is reduced under current injection to the order of 50 μM to reduce the flagellar frequency to half. The observation, illustrated in Fig. 7, that during current injection, immediately before flagellar stoppage, the flagellar frequency drops abruptly, is well in line with the interpretation that the Mg2+ content of the sperm decreases. The abrupt drop in frequency occurs when the internal Mg2+ concentration falls below 0·1 mM.

It should be noted that if Mg2+ is removed from the inside of the sperm during current injection, the Mg2+ is transported against both a chemical and an electrical gradient. An active transport system, activated by the internal potential would therefore be required. The Q10 of the time for flagellar stoppage from the start of current injection was approximately 2·6. This supports the notion that an enzymic process (a transportase) is involved in the current injection effect.

Mg2+ forms a complex with ATP. The binding constant of this complex is approximately 6 × 104 (Donaldson & Kerrick, 1975). This high binding constant means that for practical purposes all of the Mg2+ is bound as a complex with ATP whenever ATP is in excess (Storer & Cornish-Bowden, 1976). When Mg2+ is in excess over ATP, almost all of the ATP is complexed with Mg2+. To compete with the tendency of ATP to retain Mg2+ in a Mg2+-ATP complex inside of the sperm, the conjectured Mg2+ transportase should have an affinity for Mg2+ of at least an order of magnitude higher than the affinity of ATP for Mg2+. This would explain also the observation that at all Mg2+ concentrations the effectiveness of the current injection is independent of the ATP concentration (Figs. 4A, 5 A above).

It should be noted that the proposed mechanism for Mg2+ removal is very similar to the sequestering of Ca2+ by the sarcoplasmic reticulum in muscle. Ca2+ in the sarcoplasm is bound to troponin with a binding constant of approximately 6× 10ε (Ebashi, Ebashi & Kodama, 1967). The sarcoplasmic reticulum transportase is able to remove Ca2+ from the troponin and the sarcoplasm, as its binding constant for Ca2+ (7 × 106, Ebashi & Endo, 1968), is one order of magnitude higher than that of troponin.

The volume of a bull sperm is approximately 30 μm3 (= 3 × 10− 11 cm3). The removal of 1 mM Mg2+ (or 3 × 10− 17 mol Mg2+) from the sperm in 15 s requires a rate of efflux of 2 × 10− 1 − 8 mol/s. Thus with a surface area of the sperm of 140 μm2 or 1 · 4× 106 cm2, the transport rate required is of the order of 1 · 5 × 10−12 mol/cm2/s. This rate is comparable to the transport rate of Ca2+ by the sarcoplasmic reticulum, which can be estimated as 1 · 5 × 10−11 mol/cm2/s (Inesi, 1972), and the rates of active Na+ transport in giant axons of 4× 10−11 mol/cm2/s (Hodgkin, 1958) or frog skin of 20 × 10−11 mol/cm2/s (Bonting & Caravaggio, 1963).

The above considerations thus suggest that the experimental observations on the current injection effects on the flagellar activity can be explained in a simple way if an electrically activated Mg2+ transport system exists in the sperm membrane, which can reduce the Mg2+ concentration inside the sperm. The transportase would act in a way analogous to that in which the sarcoplasmic reticulum regulates the Ca2+ level in the sarcoplasm. In this latter case there is no evidence, however, of an electrical effect on the pumping action.

The mechanism for the electrical control of flagellar activity by Mg2+ transport as discussed in this paper is analogous to that found in Euglena (Nichols & Rikmenspoel, 1977, 1978). Euglena, when impaled in the presence of 1 μM gramicidin, show normal flagellar activity when only ATP, and no Mg 2+, is present in the external medium. The injection of negative current causes flagellar stoppage, analogously to that described above in bull sperm. The flagellar activity does not recover, however, after termination of the current injection. If 1 mM Mg2+ had been added to the external medium, current-induced flagellar stoppage would be followed by recovery when current injection was terminated. This experiment provides very strong evidence for a mechanism which can actively extrude Mg2+ from the Euglena.

It should be noted that the electrical control mediated by Mg2+ discussed above is different in nature from the Ca2+-mediated electrical control of ciliary motion in Paramecium (Eckert, 1972). In this latter case changes in membrane permeability to Ca2+ were involved, and no evidence for an active transport of Ca2+ was apparent.

Finally, the possible role of a control mechanism for sperm motility by Mg2+ could be questioned. It has been noted in the Introduction that ejaculated sperm do not show evidence of a control mechanism for motility. However, during storage in the epididymis sperm are generally considered immotile (Bishop, 1961, 1962) although no conclusive evidence has been presented. Upon ejaculation the sperm of most species, bull sperm among them, spontaneously start motion. A mechanism for preventing sperm motility in the epididymis, and initiating it upon ejaculation is therefore indicated. The proposed mechanism operating via the Mg2+ ion may have a physiological role here.

This investigation was supported in part by NICHD through grant HD-8752. The contribution to this work of Dr Charles Lindemann by his cooperation in the initial experiments and through discussions is gratefully acknowledged.

Bishop
,
D. W.
(
1962
).
Sperm motility
.
Physiol. Rev
.
42
,
1
59
.
Bishop
,
D. W.
(
1961
).
Biology of spermatozoa
.
In Sex and Internal Secretions
(ed.
W. C.
Young
), pp.
707
705
.
Baltimore
:
Williams & Wilkins
.
Bonting
,
S. L.
&
Caravaggio
,
L. L.
(
1963
).
Studies on sodium-potassium-activated adenosine triphosphatase. V. Correlation of enzyme activity with cation flux in six tissues
.
Archs Biochem. Biophys
.
101
,
37
46
.
Brokaw
,
C. J.
&
Simonick
,
T. F.
(
1977
).
Motility of triton demembranated sea urchin sperm flagella during digestion by trypsin
.
J. Cell Biol
,
75
,
650
665
.
Chowdbury
,
T. K.
(
1969
).
Fabrication of extremely fine glass micropipette electrodes
.
J. scient. Instrum
.
2
,
1087
1090
.
Cole
,
K. S.
&
Curtis
,
H. J.
(
1939
).
Electric impedance of the squid giant axon during activity
.
J. gen. Physiol
.
22
,
649
670
.
Donaldson
,
S. K. B.
&
Kerrick
,
W. G. L.
(
1975
).
Characterization of the effects of Mg2+ on Ca2+ and Sr2+ activated tension generation of skinned skeletal muscle fibers
.
J. gen. Physiol
.
66
,
427
444
.
Ebashi
,
S.
,
Ebashi
,
F.
&
Kodama
,
A.
(
1967
).
Troponin as the Ca2+ receptive protein in the contractile system
.
J. Biochem
.
62
,
137
138
.
Ebashi
,
S.
&
Endo
,
M.
(
1968
).
Calcium ion and muscle contraction
.
Progr. Biophys. molec. Biol
.
18
,
123
183
.
Eckert
,
R.
(
1972
).
Bioelectric control of ciliary activity
.
Science, N. Y
.
176
,
473
481
.
Gaffey
,
C. T.
&
Mullins
,
L. J.
(
1958
).
Ion fluxes during the action potential in Chara
.
J. Physiol., Lond
.
144
,
505
524
.
Gibbons
,
B. H.
&
Gibbons
,
I. R.
(
1972
).
Flagellar movement and adenosine triphosphatase activity in sea urchin sperm extracted with Triton X-10
.
J. Cell Biol
.
54
,
75
97
.
Gibbons
,
I. R.
&
Fronk
,
E.
(
1972
).
Some properties of bound and soluble dynein from sea urchin sperm flagella
.
J. Cell Biol
.
54
,
365
381
.
Gray
,
J.
(
1955
).
The movement of sea urchin spermatozoa
.
J. exp. Biol
.
32
,
775
801
.
Hodgkin
,
A. L.
(
1958
).
Ionic movements and electrical activity in giant nerve fibers
.
Proc. R. Soc. B
148
,
1
37
.
Inesi
,
G.
(
1972
).
Active transport of calcium ion in sarcoplasmic membranes
.
A. Rev. Biophys. Bioengin
.
1
,
191
210
.
Larsen
,
V. V.
,
Nielson
,
A. M. T.
,
Pape
,
L.
&
Simonson
,
L. O.
(
1971
).
The membrane potential of Ehrlich ascites tumor cells. Microelectrode measurements and their critical evaluation
.
J. Membr. Biol
.
6
,
269
288
.
Lindemann
,
C. B.
&
Gibbons
,
I. R.
(
1975
).
Adenosine triphosphate-induced motility and sliding of filaments in mammalian sperm extracted with Triton X-10
.
J. Cell Biol
.
65
,
147
162
.
Lindemann
,
C. B.
&
Rikmenspoel
,
R.
(
1971
).
Intracellular potentials in bull spermatozoa
.
J. Physiol., Lond
.
219
,
127
138
.
Lindemann
,
C. B.
&
Rikmenspoel
,
R.
(
1972
).
Sperm flagellar motion maintained by ADP
.
Expl Cell Res
.
73
,
255
259
.
Mann
,
T.
(
1964
).
Biochemistry of Semen and of the Male Reproductive Tract
.
New York
:
Wiley
.
Naitoh
,
Y.
&
Eckert
,
R.
(
1974
).
The control of ciliary activity in protozoa
.
In Cilia and Flagella
(ed.
M. A.
Sleigh
), pp.
305
352
.
London
:
Academic Press
.
Nevo
,
A.
&
Rikmenspoel
,
R.
(
1970
).
Diffusion of ATP in sperm flagella
.
J. theoret. Biol
.
26
,
11
18
.
Nichols
,
K. M.
&
Rikmenspoel
,
R.
(
1977
).
Mg2+-dependent electrical control of flagellar activity in Euglena
.
J. Cell Sci
.
23
,
211
225
.
Nichols
,
K. M.
&
Rikmenspoel
,
R.
(
1978
).
Control of flagellar motion in Chlamydomonas and Euglena by mechanical microinjection of Mg2+ and Ca’÷ and by electric current injection
.
J. Cell Sci
.
29
,
233
247
.
O’dav
,
P. M.
&
Rikmenspoel
,
R.
(
1977
).
Electrical control of flagellar activity: potentials induced by direct current injection, and temperature dependence of the electrical control of bull spermatozoon flagella
.
Biophys. J
.
17
,
267a
.
O’day
,
P. M.
,
Rikmenspoel
,
R.
&
Lindemann
,
C. B.
(
1976
).
Effects of direct current injection on the activity of bull spermatozoa
.
Biophys. J
.
16
,
119a
.
Rikmenspoel
,
R.
(
1965
).
The tail movement of bull spermatozoa. Observations and model calculations
.
Biophys. J
.
5
,
365
392
.
Rikmenspoel
,
R.
(
1957
).
An optically clear egg yolk diluent for bull spermatozoa
.
Experientia
13
,
124
126
.
Rikmenspoel
,
R.
,
Orris
,
S. E.
&
O’day
,
P. M.
(
1978
).
Ionic requirements of impaled bull spermatozoa driven by external ADP and ATP
.
Expl Cell Res
.
111
,
253
259
.
Rikmenspoel
,
R.
,
Sinton
,
S. E.
&
Janick
,
J. J.
(
1969
).
Energy conversion in bull sperm flagella
.
J. gen. Physiol
.
54
,
782
805
.
Storer
,
A. C.
&
Cornish-Bowden
,
A.
(
1976
).
Concentration of MbATP 2− and other ions in solution
.
Biochem. J
.
159
,
1
5
.