Sodium-dependent pH regulation in active sea urchin sperm

DEVELOPMENTAL

BIOLOGY

101,

425-435 (1984)

Sodium-Dependent

pH Regulation in Active Sea Urchin Sperm

THOMASBIBRING,JANE

BAXANDALL, AND CHRISTINEC.HARTER

Department of Mdecular Biology, Vanderbilt University, Nashville, Tennessee 37235 Received May 20, 1983;accepted in revised form August 22, 1983 Extracellular sodium ion is required for activation of motility and respiration in sea urchin sperm when semen is diluted in seawater. We have investigated the role of sodium ion in maintenance of sperm activity. Active sperm lose activity on transfer to sodium-free artificial seawater and can be reactivated with external Na+. Reactivation occurs in the range of Na’ concentration required for initial activation; ammonium can substitute for sodium in reactivation. Sperm withdrawn from sodium and sperm prior to activation share a characteristic morphology with straight or gently bent flagella. Activation of sperm by amines in the absence of Na+ is unstable. It is followed by a steady respiratory decline which is temporarily reversed by addition of more amine and stably reversed by addition of Na+. Measurements of intracellular pH indicate that the internal pH rises during amine activation. Internal reacidification occurs during the period of respiratory decline, and Na+ again elevates internal pH. Treatment with cyanide abolishes the reacidification, indicating that it depends on respiration. We conclude that the sodium requirement persists in active sperm; respiration-dependent production of H+ must be balanced by sodium-dependent H+ removal to maintain activity.

swered questions. In eggs, for example, the stoichiometry of sodium-hydrogen exchange has been reported to be Sea urchin sperm are inactive in semen, but respi- near 1:l (Johnson et aL, 1976; Epel, 1978a). An electroration and motility are activated on dilution of the sperm neutral exchange of sodium ion for hydrogen ion would in seawater (e.g., Gray, 1928). The activation has been reach equilibrium with equal ratios of extracellular to shown to depend on the presence of sodium ion in the intracellular concentration for sodium ion and hydrogen ion. Equilibrium would thus lead to an intracellular pH medium, and is accompanied by a sodium-dependent release of hydrogen ion from the cells (Nishioka and greater than 8, well above measured values (Shen and Cross, 1978; see also Hansbrough and Garbers, 1981) Steinhardt, 1978,1979; Grainger et c& 1979; Gillies and and by a rise in intracellular pH (Christen et aL, 1982; Deamer, 1979; Johnson and Epel, 1982). This suggests Lee et aL, 1983). Since various treatments which elevate the involvement of additional factors in pH regulation internal pH also activate sperm, it appears that the in these eggs. action of sodium ion depends on the rise in intracellular Sea urchin eggs are known to escape the sodium repH, which is more immediately responsible for acti- quirement a few minutes after activation (Chambers, vation (Christen et aL, 1982; Lee et aL, 1983). 1974; Johnson et a& 1976), but studies in which intraA similar sodium-dependent process, accompanied by cellular pH is manipulated experimentally indicate that sodium-dependent acid release and by an increase in control of internal pH remains dependent on sodium ion (Johnson and Epel, 1982; Shen, 1982). Although the intracellular pH, occurs in sea urchin eggs following fertilization (Chambers, 1974; Johnson et &, 1976; Shen first finding might suggest that intracellular pH is kept and Steinhardt, 19’78, 1979) and is responsible for the within physiological bounds by inactivation of the postulated sodium-hydrogen exchange carrier, the other activation of protein synthesis, chromosome replication, and other “late” events of the developmental program findings indicate that the situation is not this simple. (reviewed by Epel, 197813).In both eggs (Johnson et a& In the case of sperm, it has not previously been reported 1976) and sperm (Hansbrough and Garbers, 1981; see whether the sodium requirement is limited to the period also Nishioka and Cross, 1978; Schackmann and Shapiro, of initial activation, or whether it continues in active 1981; Christen et u& 1982; Lee et a.& 1983), it has been sperm. This is evidently a basic consideration in the suggested that a sodium/hydrogen exchange carrier in question of how pH is regulated in active sperm. In this paper, we will present evidence that the sodium the plasma membrane mediates the increase in intrarequirement persists in active sperm, that the cytoplasm cellular pH. is reacidified in the absence of external sodium, and that The existence of a sodium/hydrogen exchange carrier would partly explain how intracellular pH and metabolic the sperm’s metabolism itself produces most or all of rate is regulated in these cells, but would leave unan- the hydrogen ion responsible for this reaeidification. INTRODUCTION

425 6612-X66/34 $3.00 Copyright All rightn

Q 1984 by Academic Press, Inc. of reproduction in any form reserved.

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These results indicate that active sperm are in a steady Electronics, Middleton, Wise.). Measurements were on state in which metabolism produces hydrogen ion as 50 ~1 of stock sperm suspension diluted with 1.65 ml of rapidly as it is removed by the sodium-dependent system. sodium-free ASW, 50-200 ~1 of appropriate reagents The internal pH and metabolic rate of active sperm are prepared in sodium-free ASW were added as required. apparently regulated in a continuing dynamic process, Measurements of amine uptake and intracellular pH in which the sodium-dependent system of hydrogen ion were carried out after Rottenberg (1979) in connection removal and the feedback regulation of metabolism by with experiments on activation of sperm with amines. intracellular pH play vital roles. At zero time, sperm stock was diluted to l/100 in sodiumfree ASW containing activating concentrations of meMATERIALS AND METHODS thylamine or ethylamine, with 0.4 PCi [‘*C]methylHanding of spermatozoa Lytechinus pi&us and amine (New England Nuclear, Boston, Mass.) or Strong&xxntrotu.s purpuratus were obtained from Pa- 0.027 &i [‘*C]ethylamine (Research Products Internacific Bio-Marine Laboratories (Venice, Calif.). Temper- tional, Mount Prospect, Ill.), respectively. Sperm conature was regulated at 20°C in experiments with I, pit- centration in the suspension was measured by optical tus, and at 16°C in experiments with X purpuratus. All density at 350 nm and standardized against known diexperiments reported here were done with sperm of L lutions of semen and by hemocytometer counts of sperm. pictus except for the experiments recorded in Fig. 2. At appropriate times, duplicate samples of 1.4 ml were Shedding of semen was induced by intracoelomic in- centrifuged for 40 set in a Fisher microcentrifuge (Fisher jection of 0.5 1M KCl. Sperm were completely inactive Scientific, Pittsburgh, Pa.). The top part of the superin collected semen if all dilution of the semen with ex- natant was retained for measurements of external ratraneous fluids was avoided. To drain off seawater before dioactivity and pH; the lower part of the supernatant shedding, urchins were left for 30 min on absorbent was discarded. Supernatant samples of 50 ~1and pellets, paper with the aboral surface upward. Injection was which were transferred quantitatively, were placed in with a minimum of fluid, and extruded coelomic fluid scintillation vials with 10 ml of Aquasol (New England was blotted away with absorbent tissue during and after Nuclear). Radioactivity was determined in a Packard injection. Semen (containing 2-4 X lOlo sperm/ml) was Tri-carb liquid scintillation counter, Model 3320. Pellet taken up in 250-300 vol of sodium-free artificial sea- radioactivity was corrected for extracellular label, using water, and sperm were sedimented (9000 rpm, 3.5 min, data from cell volume measurements described below. Sorvall GSA rotor), resuspended in one semen-volume Zero-time measurements (measurements of intracellular of sodium-free artificial seawater (final concentration pH prior to amine activation) were made on sperm in of sperm, 60-70s of that in semen), and kept on ice up sodium-free seawater using only tracer doses of amine to several hours before use. Results were very similar (4 PM ethylamine, 66 mCi/mmol or 1 fi methylamine, over this time period except for a slight decrease in 44 mCi/mmole). Samples were allowed to equilibrate maximum respiration rate in some batches of sperm. for 15 min before processing as above. Art@ial seawater (ASW) was made up from isotonic Intracellular pH was calculated using the equation stock solutions of seawater components or activating pH, - pHi = log,, Ci/Co, where pHi and pH, are the agents, which were 1.1 M in total concentration of ions intracellular and extracellular pH and Ci and C, are the except that buffer was added without osmotic correction. intracellular and extracellular concentrations of amine. All ASW was made up calcium-free. Sodium-containing Volume determinations used in the calculations were ASW was 469 mM NaCl, 10.0 mM KCl, 25.8 mM MgClz, made on L pi&s sperm by subtraction of the space 23.0 m&f MgSOl, 2.6 mM Hepes buffer, pH 8.0. Sodium- available to sorbitol from that available to tritiated wafree ASW had the same composition with choline chlo- ter. A l/100 dilution of sperm stock was prepared in ride replacing sodium. Choline chloride was 3X recrys- sodium-free ASW containing 4 &i/ml of tritiated water tallized, from Sigma Chemical Company (St. Louis, MO.). (0.25 mCi/gm, New England Nuclear). The sperm conIn some experiments, 99% choline chloride from the centration was determined as above. The suspension same source was used, with no difference in the results. was left to equilibrate for 5-20 min at 2O“C or l-2 hr Sodium-containing and sodium-free ASW were mixed at 4°C (Lee et al, 1983) and 0.2 &i/ml of [14C]sorbitol to obtain seawater with the specified concentrations of (324 mCi/mmole, New England Nuclear) was added. Afsodium ion. Other components were added to sodium- ter 2 min at 2O”C, sperm and supernatant were collected free ASW from isotonic stocks to give the specified con- and prepared for radioactivity determination as above, centrations. and subjected to two-channel counting for i*C and triRes?yirometrll was carried out by continuous recording tium. A volume of 9.2 pm3 was obtained, in reasonable in the stirred, water-cooled cell of a Gilson 5/6H Oxy- agreement with the volume of 8 pma reported by Lee et graph fitted with a Clark electrode (Gilson Medical al. (1933) for sperm of the same species.

BIBRING, BAXANDALL, AND HARTER

pH Regulation in Active Sperm

427

on reversed sperm and sperm not previously activated show indistinguishable effects of Na+ in this concenAbsence of the Acrosome Reacticm under the tration range. Experimental Conditicms Used Third, addition of lo-30 mMammonium (pH 8) to the All media used in this study were made up calcium- medium activates vigorous motility in sperm deactivated free to avoid spontaneous occurrence of the acrosome by transfer to Nit+-free ASW, as it does in sperm not reaction and other complications (Lee et ah, 1983) from previously activated. The accepted action of ammonium the presence of calcium. Occurrence of the acrosome is to raise the intracellular pH by diffusion of the unreaction was monitored by dark-field microscopy, which charged basic form into the cell (Thomas, 19’74;Aickin revealed both extension of the acrosomal filament and and Thomas, 1977; Boron and De Weer, 1976, Boron, a morphological change in the acrosomal vesicle in sperm 1977; Roos and Boron, 1981; Shen and Steinhardt, 1978; in which the acrosome reaction was induced by exposure Grainger et aL, 1979; Johnson and Epel, 1982; Christen to egg jelly (data to be presented in more detail else- et al, 1982;Lee et aL, 1983), and the ability of ammonium where). In sperm in calcium-free ASW activated by 25 and amines to activate sperm in the absence of Na+ is mM Naf or by ammonium up to 20 mM, no acrosome- evidence that Na+, also, acts by elevating internal pH (Nishioka and Cross, 1978; Christen et al, 1982; Lee et reacted sperm were detected by these means. al, 1983). Reversibility of Sodium-Dependent Activation It is reasonable to conclude that Na+ plays the same role in the maintenance of sperm activity as in initiation of Sea Urchin Sperm of sperm activity, and that continuous removal of H+ To determine whether sperm, like eggs, escape the from sperm is required to maintain the active state. sodium requirement for metabolic activity, sperm of L. p&w which had previously been washed in sodium-free Instability of Amine Activation ASW were activated in ASW containing 25 mM Na+. As already mentioned, sperm can be activated in the At 2, 5, and 10 min after activation, the sperm were collected by centrifugation and resuspended in a large absence of sodium ion by amines; however, we have volume of sodium-free ASW (at least 300X the volume consistently observed that activation by amines is unof washed sperm used initially). This resulted in com- stable. Figure 2a shows the time-course of respiration plete loss of motility and respiration, while motility and in sperm activated by various concentrations of amrespiration were unaffected in controls which were cen- monium at pH 8.0. After activation with 10 mM amtrifuged and resuspended in Na+-containing ASW. monium, the respiration rate initially equals the maxSperm deactivated by removal of Na+ can be fully reac- imum rate obtainable with Na+, but respiration begins tivated by addition of Na+, provided that the dilution to fall within a few minutes, and continues to fall for of sperm with Na+-free medium has not been much the rest of the measurement. For ammonium concengreater than 1:300 (the addition of 0.3 mM Na+ to the trations above 10 mM, the period of maximal respiration Na+-free ASW used protects against irreversible effects lengthens as the ammonium concentration increases; from high dilution) and the sperm have not been left for ammonium concentrations below 10 mM, the resin low-Na+ medium longer than approximately 2 hr. piration rate is below maximum initially and immeSeveral observations indicate that the state of sperm diately declines. We have repeated these measurements deactivated by transfer to Na+-free medium is identical using methylamine, ethylamine, or diethylamine as the to the state of sperm prior to activation. First, deac- activating amine. The effects of given concentrations of tivated sperm have the same distinctive morphology as amine and the time-course of inactivation are similar sperm prior to activation (Fig. 1). In both cases, the for all amines tested. tails of the sperm are straight or slightly bent; a sharp Christen et al. (1982) have previously reported resbend, when present, is limited to a short region behind pirometric results on sperm activated with ammonium. the head. This morphology suggests that both groups Their data are similar to ours except that 10 mM amof sperm are blocked in the same specific stage of the monium results in several minutes of stable respiration. dynein ATPase cycle (see Discussion). Second, the dose This difference is probably due to a difference in sodium of Na+ required for activation is similar or identical for concentration in the media used, since Christen et al. sperm prior to activation and deactivated sperm. In cal- diluted semen 1:200 in sodium-free medium for their cium-free seawater, which was used in all these exper- study, while a considerably greater dilution was used iments, initial activation of sperm occurs feebly in l-4 in the work reported here. These results are consistent with the regulation of mM Na+, but strongly in 6 mM Na+. The same result is obtained by respirometry (data not shown) or by ob- respiration rate by intracellular pH. Concentrations of servation of motility. Parallel observations of motility ammonium below 10 mM evidently do not raise the inRESULTS

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DEVELOPMENTALBIOLOGY

VOLUME101,1984

FIG. 1. The characteristic morphology of inactive sperm in sodium-free seawater. Phase-contrast micrograph of living sperm of L pictus after dilution of semen in sodium-free seawater. Flagella are straight or slightly bent. A sharper bend, when present, is limited to the extreme anterior region of the flagellum. Sperm activated by transfer to normal seawater and deactivated by transfer to sodium-free seawater have an identical morphology (not shown).

10

mM

NH4+

-4

5

a

25

mM

min

Na+

FIG. 2. (a) Instability of respiration after ammonium-activation of sperm in sodium-free seawater. Sperm from semen of 5: purpuratus were washed in sodium-free seawater and aliquots, resuspended in sodium-free seawater, were placed in the Oxygraph cell. Sodium chloride or ammonium chloride (pH adjusted to 8.0 with KOH) was added at the arrow. The respirometer traces obtained in different runs have been superimposed for comparison. The trace for 25 mM sodium represents stable, maximal respiration. Ammonium concentrations below 10 mM give respiration which is unstable and submaximal. Ammonium concentrations of 10 mM or greater give maximal respiration initially, and the period of maximal activation is progressively longer for higher ammonium concentrations. Respiration eventually declines in all cases; the decline continues steadily throughout the period of measurement. (b) Sodium and ammonium reverse the spontaneous inactivation of ammonium-activated sperm. Sperm were prepared for respirometry as in (a). Aliquots of the same sample were used for the two runs shown. The respirometer traces obtained are displayed with a vertical offset. At the first arrow, ammonium was added to both samples to a concentration of 10 n&f. At the second arrow, a second dose of ammonium, equal to the first, was added (upper trace), or sodium was added to a concentration of 25 mM (lower trace). Both treatments restore maximal respiration. Ammonium activation is again unstable, while sodium activates stably.

BIBRING, BAXANDALL, AND HARTER

tracellular pH sufficiently for maximum respiration, while ammonium concentrations of 10 mM or greater allow maximum respiration. For all concentrations, however, respiration sooner or later declines, apparently due to a progressive intracellular acidification. For concentrations below 10 mM, the decline is immediate, while ammonium concentrations above 10 mM apparently raise the internal pH above that required for maximum respiration, allowing some reacidification to occur before the respiration rate is affected. If the decline in respiration rate is due to intracellular acidification, then addition of a second dose of amine during the inactivation phase should reverse the inactivation by raising the intracellular pH. This is the case, as shown in Fig. 2b, as might be expected, the activation is again unstable. The ability of amines to reverse inactivation rules out the possibility that inactivation is caused by toxic secondary effects of amines. Sperm which have lost activity in the presence of amine can also be rescued by Na+. An activating concentration (25 mM) of Na+, added to the medium of amine-activated sperm during the period of respiratory decline, gives an increase in respiration, usually to the saturated level (Fig. 2b). After addition of Na+, respiration is stable. A nonactivating concentration (1 mM) of Na+ has no discernible effect. The decay of respiration in amine-activated sperm in the absence of Na+, and the rescue of respiration by Na+, provide further evidence that sperm do not escape the Na+ requirement after activation, and support the interpretation that Na+ continues to mediate the removal of intracellular hydrogen ion in active sperm. Direct Measurements of Intracelhlar

pH Chnges

We have tested directly the interpretation that the intracellular pH of sperm declines during the period of decreasing respiration after activation by amines, and that it rises again on addition of Na+. Amine accumulation is a measure of intracellular pH (reviewed by Gillies and Deamer, 1979; Rottenberg, 1979; Roos and Boron, 1981; see also Roos and Keifer, 1982; Christen et aL, 1982; Lee et &, 1983). Tracer doses of amines are usually used in such measurements; the experiments described here call for interventive doses, but this does not affect the measurements themselves. Radiolabeled ethylamine was used to measure accumulation of ethylamine, and radiolabeled methylamine to measure accumulation of methylamine. The uptake of ethylamine, when present in the medium at 20 mM, is plotted in Fig. 3. Results for methylamine are quite similar. The uptake is evidently biphasic. Rapid uptake accompanies activation, and a

pH Regulation in Active Sperm

60

429 Na’ v R

r

10

20 Time

30

40

(min)

FIG. 3. Uptake of ethylamine during activation of sperm with ethylamine and the subsequent period of respiratory decline. Sperm of L pi&us were used in this experiment and all later experiments. Radiolabeled ethylamine (20 mM) was added to sperm in sodium-free seawater at zero time, and the uptake of ethylamine per gram wet weight of sperm (2-4 X 10” sperm) is plotted. Rapid uptake of ethylamine accompanies activation. Uptake continues at a slower but significant rate during the period of respiratory decline; this is interpreted as reflecting the titration of basic amine by H+ released within the cells. Sodium, added at the arrow to a concentration of 35 m&f, drives the amine accumulated during respiratory decline back out of the cell.

slow but substantial accumulation continues during the period of respiratory decline. This is the expected result if large amounts of H+ are released in the cytoplasm of respiring sperm, since cytoplasmic Hf would titrate basic amine, and basic amine would enter the sperm to maintain diffusion equilibrium. Addition of Na+ drives the amine accumulated during respiratory decline back out of the cell, presumably by reversing the acidification. Intracellular pH, calculated from uptake measurements, is shown in Fig. 4 for sperm activated by 10 mM methylamine or by 20 mM ethylamine. In each case, there is a pH increase associated with amine activation, a pH decrease during inactivation, and a pH rise on addition of Na+ to a level consistent with maximal activation. The absolute values of pH obtained in these measurements are somewhat lower than those obtained by Lee et al. (1983) on sperm of the same species. We do not know the reason for this discrepancy (see Discussion for a consideration of uncertainties of these measurements). By simultaneous measurement of respiration rate and amine accumulation before and after amine activation, during the period of respiratory decline, and after reactivation with Na+, plots of respiration rate vs intracellular pH can be constructed. The system is very favorable for this purpose, because of the continuity in

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DEVELOPMENTAL BIOLOGY VOLUMElOl.1984

tifact of measurement, nor artifactually the amines (see Discussion).

produced by

Stead;ll-State pH Regulation in Active Sperm

6.29

’

I 10

I Time

I 20

I

I 30

I

I 40

(min)

FIG. 4. Changes in intracellular pH during activation of sperm by aminea, the subsequent respiratory decline, and reactivation by sodium. Radiolabeled methylamine (10 m&f; circles) or ethylamine (20 m&f; squares) were added at zero time to sperm in sodium-free seawater. Intracellular pH was calculated from measurements of amine uptake (zero-time pH values were obtained in measurements on sperm in sodium-free seawater, using tracer doses of amines). Sodium (25 mil4; open symbols) was added at the arrows to aliquots of the amineactivated sperm. Intracellular pH rises sharply during activation with amines, and falls gradually during the period of respiratory decline toward the value characteristic of inactive sperm. Addition of sodium rapidly and stably restores the pH to values characteristic of active sperm.

values of internal pH and respiration rate during the period of respiratory decline. The plots obtained are shown in Fig. 5. They are internally consistent, and there is good agreement between plots obtained in separate experiments using different amines. These plots fully support the proposition that the changes in respiration rate in these experiments reflect changes in intracellular pH. The transition of respiration with pH is extremely sharp, indicative of a highly cooperative process. The Source of Cytoplasmic Hydrogen Ion in Active Sperm To determine whether respiration itself might be the source of the hydrogen ion which accumulates in active sperm in the absence of Na+, we measured internal pH in sperm exposed to amines in the presence of cyanide. In the presence of 0.2 mMCN-, respiration is completely blocked (Fig. 6a). The initial elevation of pH, which in unblocked sperm results in activation, is undiminished, but the subsequent acidification is sharply depressed, and probably entirely absent (Fig. 6b). This result indicates that the acidification of cytoplasm in active sperm is a consequence of respiration itself, and that passive influx from the medium (driven by the membrane potential, but otherwise passive) is not an important source of H+ ions. The effect of cyanide further argues that the observed acidification is neither an ar-

The above results indicate that both respiration-dependent release and sodium-dependent removal of intracellular hydrogen ion occur continually in active sperm. It is implicit in these findings that intracellular pH in active sperm is determined by a steady state of hydrogen ion production and removal, since hydrogen ion will tend to the level where its production and removal occur at equal rates. This steady state is diagrammed in Fig. 7. The “feedback loop” by which intracellular pH controls respiration rate is also indicated, and some possible side branches of the steady state are drawn. DISCUSSION

We have shown that extracellular sodium ion is required not only for initial activation of sperm, but also for maintenance of sperm activity (motility and respiration). A number of results indicate that the function of sodium ion in active sperm, as in initial activation, is the removal of intracellular hydrogen ion. Similar or identical concentrations of sodium ion are required for initial activation of sperm and for reactivation after withdrawal of sodium ion. Ammonium, which raises the

a,

I

II

’

I

/ I

6.2

6.4 Internal

pH

FIG.5. Relationship of respiration rate to intracellular pH in sperm activated with amines. Sperm in sodium-free seawater were activated with methylamine (10 mM; squares) or ethylamine (20 mM; circles) containing radiolabel, and divided into two groups. Respirometric measurements were carried out on one group and determinations of intracellular pH were carried out on the other. Measurements were carried out during the period of respiratory decline and after reactivation of aliquota of the sperm with Na+. Measurementa of respiration rate and internal pH were also carried out on sperm in sodium-free seawater prior to activation. The respiration rate at various times was plotted against the intracellular pH at the same time. The resulting curves show a smooth relationship between respiration rate and intracellular pH, and the curves from separate experiment-s coincide well. A sharp transition of respiration rate with pH is indicated.

BIBRINC, BAXANDALL, AND HARTER

pH Regulation

in Active Sperm

431

+CN-

I

E ZL

CL

-

5 min

2

; 6.4 a -c

6.2 b

I

I

I

I

10

20 Time

I

I

30

(min)

FIG. 6(a). Blockage of sperm respiration by 0.2 mil4 cyanide. Sodium-free seawater without (lower trace) or with (upper trace) 0.2 mM cyanide was placed in the Oxygraph cell. At the first arrow, aliquots of the same sperm used for internal pH measurements (b) were added. The dip in oxygen level on addition of sperm probably reflects a low oxygen level in the added sample. At the second arrow, 20 mA4 ethylamine was added. Cyanide-blocked sperm were unresponsive to ethylamine, while respiration in controls was activated normally. (b) Cyanide blocks the internal reacidification of amine-treated sperm. Aliquots of sperm were placed in sodium-free seawater without (solid symbols) or with (open symbols) 0.2 mM cyanide. At zero time, 20 mM ethylamine containing radiolabel was added, and internal pH was determined by measurement of ethylamine uptake. A comparable elevation of internal pH occurs in controls and in cyanide-blocked sperm on exposure to ethylamine. Reacidification then takes place in controls, but is sharply reduced or absent in blocked sperm.

Na+

Na/H

Na/K

Exchange

ATPase

FIG. ‘7. Steady-state pH regulation in active sperm. The diagram shows the respiration-dependent release and sodium-dependent removal of intracellular hydrogen ion in active sperm. Intracellular H+ reaches the level at which its production and removal occur at equal rates. There is a negative feedback between H+ and respiration; high H+ inhibits ATP utilization (Christen et al, 1969) and directly or indirectly inhibits respiration, thereby decreasing H+ production. For reasons given in the discussion, respiration-dependent H+ is viewed as deriving from carbon dioxide, and bicarbonate ion is viewed as leaving the cell by a separate route. Other likely branches of the steady state are drawn in the lower part of the figure: Na+ enters the cell by exchange with hydrogen ion and is removed again by Na+/K+ active transport; K+ taken up by this route is viewed as leaving by “leak” pathways.

intracellular pH, can substitute for sodium ion in either case. Sperm prior to activation and sperm withdrawn from sodium have the same characteristic morphology in which the tails are only slightly bent, often extending straight back from the head. The available evidence suggests that the straighttailed morphology reflects a specific blockage of the dynein ATPase cycle in the stage at which the dynein arms are detached from the adjacent B microtubules (relaxed stage). The action of symmetrically arranged elastic elements is expected to straighten the flagella when the arms are detached (Gibbons and Gibbons, 1974). Demembranated flagella in which the dynein ATPase is blocked by vanadate assume a straight morphology (Gibbons et aL, 1978; Okuno, 1981). These flagella have the low degree of stiffness expected from a condition in which the arms are detached (Okuno, 1981), and in trypsin-treated flagella in which connections between doublets appear to be maintained primarily by arm attachments (Summers and Gibbons, 1973), the doublets separate from each other when the dynein ATPase is blocked by vanadate (Sale and Gibbons, 1979). Under conditions of limiting availability of Mg-ATP, flagella are also straight (Gibbons and Gibbons, 1974) and have a low degree of stiffness (Okuno and Hiramoto, 1979; Okuno, 1981). Concentrations of Mg-ATP which straighten the flagella are insufficient to produce fla-

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DEVELOPMENTAL BIOLOGY VOLUME101.1984

gellar beating (Gibbons and Gibbons, 1974), but sufficient to dissociate dynein from B-tubules in vitro (Takahashi and Tonomura, 1978). Available evidence further suggests that blockage in the relaxed stage is characteristic of sperm having a low internal pH. In addition to sperm blocked by the absence of sodium, sperm blocked by a low-pH medium (Goldstein, 1979) or a low-pH medium in the presence of CO, (Okuno and Hiramoto, 1979) have straight flagella. The expectation that these sperm have a low internal pH has been confirmed by measurements (Christen et a& 1982). In the case of sperm blocked by low pH and COz, the flagella have been shown to have a low degree of stiffness, similar to that of flagella blocked by vanadate (Okuno and Hiramoto, 1979). The behavior of sperm activated with amines further supports the conclusion that external sodium is required in active sperm for maintenance of a high intracellular pH. Respiration falls steadily in these sperm, but is restored by treatments expected to raise the internal pH: amines reactivate transiently, and sodium reactivates stably. Direct measurements of internal pH by determination of amine accumulation confirm that the cell interior is reacidified during the period of respiratory decline, and that addition of sodium reverses the acidification. Measurements of internal pH by amine accumulation are subject to some uncertainties. Calculation of pH from amine uptake requires the amine to be at titrationdiffusion equilibrium throughout the measurement, while the work presented here requires dynamic measurements. The measurements should nevertheless be useful as approximations. The acidification we observe continues over a time span of a half hour. Tracer doses of the amines used equilibrate in approximately 15 min, (Christen et al, 1982; Lee et u& 1983), and the use of interventive rather than tracer doses of amines in our experiments speeds approach to equilibrium, since the resulting intracellular pH changes combine with diffusion movement of the amine to drive the system toward equilibrium. Our results specifically indicate that the observed acidification is not an artifact of measurement (resulting from amine accumulation while uncharged amine is approaching distribution equilibrium). The uptake curve is biphasic, and does not appear to level off. Cyanide, which should not affect the course of uptake of uncharged amines, does not affect the initial uptake connected with elevation of internal pH, but suppresses the acidification. The data on internal pH in the presence of cyanide (Fig. 6b) in fact suggest that uptake equilibrium is reached within minutes under the conditions used. A further uncertainty of pH measurements based on amine accumulation is the possibility that the acid form

as well as the basic form penetrates the cell, distorting the measurement. This raises the possibility that the observed acidification, while real, is an experimental artifact caused by gradual entry of the charged form of the amine. A number of considerations argue strongly against this possibility. A comparable slow phase of amine uptake has not been observed in other studies (Christen et al, 1982; Lee et aL, 1983). One would not expect the rates of uptake of different charged amines to be the same, yet there is no reproducible difference in the time-course of inactivation with different amines. Moreover, the occurrence of an artifactual reacidification after activation with amines does not explain the inactivation which occurs, in the absence of amines, when sodium is removed from the medium. Finally, the reacidification is blocked by cyanide, which should not affect the rates of uptake of charged amines. The absolute values of internal pH obtained in our measurements are unexpectedly low, and below those obtained by Lee et aL (1983) using methylamine accumulation with sperm of the same species. We do not know the reason for this discrepancy, but it is important to recognize that the measurement of changes in internal pH is expected, on both theoretical and experimental grounds, to be more accurate than the measurement of absolute values. This is because intracellular binding of amine, on which conclusive information is unavailable, can distort the measured absolute values but is expected to have relatively little effect on measured changes (Lee et uL, 1983), and because errors in the measurement of intracellular volume can significantly affect absolute values, but have no effect on measured changes. The pH change on activation obtained in our measurements is comparable to that obtained in other studies (Christen et uL, 1982; Lee et uL, 1983). Since our interpretations depend only on pH changes, and do not depend on their exact values, the pH measurements should be adequate for their intended purpose. We conclude from its blockage by cyanide that intracellular acid release in active sperm depends on respiration. Does this acid derive from COz? Since COz accumulates as a respiratory product, and exists largely as bicarbonate and H+ at the intracellular pH, any significant steady-state efflux of bicarbonate through its own exit route would leave H+ to be removed by the sodium-dependent system or to accumulate in the cell (see Fig. 7). Indirect evidence strongly supports this possibility. In the presence of sodium, the respirationdependent intracellular H+ is released from the cell (Fig. 4). It should therefore correspond to the acid known to be released into the medium by respiring sperm (the “slow acid release” of Nishioka and Cross (1978), which is respiration-dependent). Data of Mohri and Horiuchi (1961) indicate that this acid does not accumulate in the

BIBRING, BAXANDALL, AND HARTER

medium when carbon dioxide is continuously removed. However, the experiment was not specifically designed to test the nature of the acid released by sperm, and interpretation of the result requires caution (Holland and Gould-Somero, 1982). We are currently reinvestigating this question; the results to date confirm that the respiratory acid is carbon dioxide. The plot of internal pH against time (Fig. 4) may appear to contradict the inference that the acid released in active sperm is a direct result of respiration, in that internal pH does not decrease most rapidly immediately after activation, when respiration is greatest. Intracellular compartmentation could be responsible for a delay between respiratory acid production and a decline in measured internal pH. The respiratory acid could at first be released in the relatively alkaline mitochondrial compartment, from which the amine is largely excluded, and might only gradually achieve a distribution to which amine uptake is more responsive. Because of this possibility, and the other uncertainties in internal pH measurements, we suggest that greater weight should be given to other evidence concerning the source of the acid: its production is supressed by cyanide, and it appears to be carbon dioxide. Lee et aL (1983) have also observed (in sperm of L. pictus but not of S. purpuratus) a respiration-dependent internal reacidification following activation. However, this reacidification occurred under conditions quite different from those used here, and apparently results from a different mechanism. It occurred in sodium-activated sperm, required the presence of calcium in the medium, and was accompanied by a large uptake of calcium. In agreement with these results, we find that respiration is unstable in sodium-activated sperm of L pictus when calcium is present in the medium (unpublished data), but stable when calcium is absent (Figs. 2a, b). Calcium was omitted from all media used in the present study, and the unstable respiration and accompanying reacidification reported here appear to occur only in the absence of sodium (in amine-activated sperm) and are independent of calcium. Our results and interpretations are summarized in Fig. 7, which indicates that active sperm come to a steady state of respiration-dependent production and sodiumdependent removal of intracellular hydrogen ion. In this context, the inhibitory effect of internal H+ on respiration acts as a negative feedback loop. If for any reason respiration-dependent production of hydrogen ion exceeds the rate of sodium-dependent H+ removal, then H+ accumulation sharply inhibits respiration, and the resulting reduction in H+ production is a major factor in rebalancing the system. The new steady state which results is characterized by a lower respiration rate, and because of the extreme sensitivity of respiration rate

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to pH (Fig. 5), regulation over the entire range of respiration rates occurs within a narrow range of intracellular pH. This apparently explains the inactivation of sperm in the absence of external sodium. Respiration is in turn a major factor in the regulation of intracellular PH. Ultimate control over internal pH and respiration may be exerted by the sodium-dependent system of H+ removal, which is itself subject to external regulation (Hansbrough and Garbers, 1981). As indicated in Fig. 7, the inhibition of respiration by low intracellular pH is not necessarily direct. Christen et al. (1983) have recently shown that ATP levels are high, but ATP utilization is low, in sea urchin sperm with a low intracellular pH, suggesting a direct effect of low intracellular pH on ATP utilization (assumed to be mainly dynein ATPase activity). Respiration is also inhibited at low intracellular pH, and the unavailability of ADP as a respiratory substrate does not account for this, since uncoupled respiration is also inhibited. However, it is not yet known whether the inhibition of respiration is a direct effect of internal pH or the result of a regulatory pathway driven by ATP/ADP balance. In our view, inactive sperm in sodium-free seawater are in essentially the same physiological state as active sperm, except that in the absence of effective means of H+ removal, respiration proceeds at a very low rate, and systems such as motility that are regulated within the same pH range are inhibited. The rapid release of acid to the medium on activation of sperm (Nishioka and Cross, 1978) apparently reflects the relaxation of the steady state of H+ production and removal from one condition to another when an effective means for H+ removal is supplied. It is tempting to consider a uniform interpretation of the inactivity of sperm in sodium-free seawater and the inactivity of sperm in semen; sperm in semen, like sperm in sodium-free seawater, release acid to the medium when diluted into seawater (Nishioka and Cross, 1978). Does the inactivity of sperm in semen result from self-inhibition when removal of intracellular H+ is inadequate? Although seminal fluid contains sodium, the efficiency of H+ removal must be reduced in semen by the crowding of sperm, since accumulation of H+ in the medium would tend to reverse the further efflux of H+. With bicarbonate present in the medium, COz would also accumulate, and tend to reverse any efflux of H+ directly as COz, an aspect of inhibition not present in dilute sperm in sodium-free seawater. Both extracellular H+ and extracellular COzare known inhibitors of sperm activity, and have long been considered to play a role in the inactivation of sperm in semen (Mohri and Yasumasu, 1963; Ohtake, 1976; Kopf et aL, 1979; Hansbrough and Garbers, 1981; Christen et aL, 1982). The view that crowding itself is responsible for the

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inactivity of sperm in semen has direct support, in that they can be activated by dilution even in seminal fluid (Gray, 1928; Rothschild, 1948). Crowding, like removal of sodium, can also reverse the activity of sperm in seawater (our unpublished results). If active sperm in seawater are packed into a pellet by centrifugation, they lose activity and remain viable for several days in the refrigerator (this is in fact an effective procedure for storage of sperm). It seems that perturbation of the steady state of H+ removal by crowding must be a significant factor in the inactivity of sperm in semen. Interpretation of the results presented here has depended on the view that respiration rate in sea urchin sperm is regulated by intracellular pH. At the same time, the data are entirely consistent with this view, and can be considered to support it. The activation of respiration with amines, the ensuing inactivation, and the reactivation of respiration by Na+ are all accompanied by the expected changes in intracellular pH. The ability of amines to reactivate respiration is also according to expectation, as is the delay in respiratory inactivation when sperm are exposed to doses of amines higher than those required to give maximal respiration. Apparently these doses take the intracellular pH above that required for maximal respiration, and some accumulation of intracellular Hf occurs before respiration falls again. The clearest expression of the correlation between pH and respiration rate is the plot of respiration rate against intracellular pH, based on measurements made during inactivation and reactivation of sperm in the presence of amines. The plot traces out a clean curve, and two curves made at different times and using different amines coincide closely. This work was supported by NSF Grant PCM 8105681, in part by Grant BSRG S-07 RR07201from NIH, and by a grant from the Natural Science Fund, Vanderbilt University. A preliminary report on this work has appeared (Bibring and Baxandall, 1982). REFERENCES AICKIN, C. C., and THOMAS,R. C. (1977). An investigation of the ionic mechanism of intracellular pH regulation in mouse soleus muscle fibers. J. Physiol (London) 273,295-316. BIBRING,T., and BAXANDALL,J. (1982). Sodium-dependent pH regulation in activated sea urchin sperm. J. CeUBioL 95, (2, Pt. 2). 164a. BORON, W. F. (1977). Intracellular pH transients in giant barnacle muscle fibers. Amer. J. Physid 233, C61-C73. BORON,W. F., and DE WEER, P. (1976). Intracellular pH transients in squid giant axon caused by CO,, NH8, and metabolic inhibitors. J. Gen PhysioL 67, 91-112. CHAMBERS, E. L. (1974).Na is essential for activation of the inseminated sea urchin egg. J. Exp. ZooL 197, 149-157. CHRISTEN,R., SCHAaIIIANN, R. W., and SHAPIRO,B. M. (1982).Elevation of the intracellular pH activates respiration and motility of sperm of the sea urchin, Stwngylocatrotus purpuratus J, Bid Ch 257, 1481-1490.

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CHRISTEN, R., SCHACKMANN, R. W., and SHAPIRO, B. M. (1983). Me-

tabolism of sea urchin sperm. Interrelationships between intracellular pH, ATPase activity, and mitochondrial respiration. J. Bid Chewy 258, 5392-5399. EPEL, D. (1978a). Intracellular pH and activation of the sea urchin egg at fertilization. In “Cell Reproduction” (E. R. Dirksen, D. Prescott, and D. F. Fox, eds.), pp. 367-378. Academic Press, New York. EPEL, D. (1978b). Mechanisms of activation of sperm and egg during fertilization of sea urchin gametes. Curr. Top. LJeu. Bid 12, 185246. GIBBONS,B. H., and GIBBONS,I. R. (1974). Properties of flagellar “rigor waves” formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm. J. Cell Bid 63,970-985. GIBBONS,I. R., COSSON,M. P., EVANS, J. A., GIBBONS,B. H., HOUCK, B., MARTINSON,K. H., SALE, W. S., and TANG, W-J. Y. (1978). Potent inhibition of dynein adenosinetriphosphatase and of the motility of cilia and sperm flagella by vanadate. Proc NatL Ad Sci USA 75,2220-2224. GILLIES, R. J., and DEAMER,D. W. (1979). Intracellular pH: Methods and applications. Curr. Top. Bioenerg. 9, 63-87. GOLDSTEIN,S. F. (1979).Starting transients in sea urchin sperm flagella. J. Cell Bid 80.61-68. GRAINGER, J. L., WINKLER,M. M., SHEN, S. S., and STEINHARDT,R. A.

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JOHNSON,J. D., EPEL, D., and PAUL, M. (1976). Intracellular pH and activation of sea urchin eggs after fertilization. Nature (London) 262,661-664. KOPF, G. S., TUBB, D. J., and GARBERS,D. L. (1979). Activation of sperm respiration by a low molecular weight egg factor and by 8bromoguanosine 3’,5’ monophosphate. J. BioL Chem. 254,8554-8%X LEE, H. C., JOHNSON,C. H., and EPEL, D. (1983). Changes in internal pH associated with initiation of motility and acrosome reaction of sea urchin sperm. Dev. BioL 95, 31-45. MOHRI,H., and HORIUCHI,K. (1961). Studies on the respiration of seaurchin spermatozoa. III. Respiratory quotient. J. Exp. Bid 38,249257. MOHRI,H., and YASUMASU,I. (1963). Studies on the respiration of sea urchin spermatozoa. V. The effect of Pcoo.J.Exp. BioL 40,573-586. NISHIOKA, D., and CROSS,N. (1978). The role of external sodium in sea urchin fertilization. In “Cell Reproduction” (E. R. Dirksen, D. Prescott, and D. F. Fox, eds.), pp. 403-413. Academic Press, New York. OHTAKE,H. (1976). Respiratory behaviour of sea urchin spermatozoa. I. Effect of pH and egg water on the respiratory rate. J. Exp. ZooL K&303-312. OKWO, M. (1981). Inhibition and relaxation of sperm flagella by vanadate. J. Cell Bid 85,721-725. OKmo, M., and HIRAMoTO, Y. (1979). Direct measurements of the stiffness of echinoderm sperm flagella. J. Eq Bid 79.235-243. ROOS,A., and BORON,W. F. (1981). Intracellular pH. Physiol R~. 61, 296-434. Roe% A., and KEIFER. D. W. (1982). Estimation of intracellular pH from distribution of weak electrofytes. In “Intracellular pH: Its

BIBRINC, BAXANDALL, AND HARTER Measurement, Regulation, and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamer, eds.), pp. 55-59. Liss, New York. ROTHSCHILD,LORD (1948). The physiology of sea urchin spermatozoa. Lack of movement in semen. J. Exp. Biol 25,344-352. ROT~ENBERG, H. (1979). The measurement of membrane potential and pH in cells, organelles and vesicles. In “Methods in Enzymology” (S. Fleischer and L. Packer, eds.) Vol. 55, pp. 547-569. Academic Press. New York. SALE, W. S., and GIBBONS,I. R. (1979). Study of the mechanism of vanadate inhibition of the dynein cross-bridge cycle in sea urchin sperm flagella. J. CeU Bid 82, 291-298. SCHACKMANN,R. W., and SHAPIRO,B. M. (1981). A partial sequence of ionic changes associated with the acrosome reaction of Stromg¢rotua purpumtus. Lkv. Bid 81.145-154. SHEN, S. S. (1982). The effect of external ions on pHi in sea urchin eggs. In “Intracellular pH: Its Measurement, Regulation, and Uti-

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lization in Cellular Function” (R. Nuccitelli and D. W. Deamer, eds.), pp. 269-282. Liss, New York. SHEN, S. S., and STEINHARDT,R. A. (1978). Direct measurement of intracellular pH during metabolic derepression of the sea urchin egg. Nature (London) 272.253254. SHEN, S. S., and STEINHARDT,R. A. (1979). Intracellular pH and the sodium requirement at fertilization. Nature (London) 282,87-89. SUMMERS,K. E., and GIBBONS,I. R., (1973). Effects of trypsin digestion on flagellar structures and their relationship to motility. J CeU Bid 58,618-629. TAKAHASHI, M., and TONOMURA,Y. (1978). Binding of 30s dynein with the B-tubule of the outer doublet of axonemes from Tetm&nerza &or-m& and adenosine-triphosphate-induced dissociation of the complex. J. Biochea (Tokyo) 84,1339-1355. THOMAS,R. C. (1974). Intracellular pH of snail neurones measured with a new pH-sensitive glass micro-electrode. J. PhgsioL (Lundon) 238, 159-180.