A volatile inhibitor immobilizes sea urchin sperm in semen by depressing the intracellular pH

DEVELOPMENTAL

BIOLOGY

98,493-501

(1983)

A Volatile Inhibitor Immobilizes Sea Urchin Sperm in Semen by Depressing the Intracellular pH CARL HIRSCHIE JOHNSON,**’ DAVID L. CLAPPER,* MATTHEW M. WINKLER,t HON CHEUNG LEE,* AND DAVID EPEL*~’ *Hopkins Marine Station, Department of Biological Sciewes of Starlford University, P&c Grove, Califiia 95950; TDepartment of Biological Chemistry, School of Medicine, University of California, Davis, Cal$ornia 95616; and *Deportment of Physiology, 6-246 Millard Hall, University of Minnesota, Minneapolis, Minnesota 55.455 Received August 11, 1982; accepted in revised

form March

23, 1986

Sea urchin spermatozoa are normally immotile in semen, but motility can be initiated by increasing gas flow over the semen-for example, by blowing Ns gas over a thin layer of semen. This result indicates that sperm motility is not O2 limited and suggests that seminal fluid contains a volatile inhibitor of motility which is responsible for the paralysis of sperm in semen. This inhibitor might be carbon dioxide, which reversibly immobilizes sperm. 31P-NMR measurements of pH show that the sperm intracellular pH (pHi) increases by 0.36 pH unit upon dilution of semen into seawater. Since previous studies have shown that this magnitude of pH increase is sufficient to trigger sperm motility, we suggest that the volatile inhibitor is inhibiting sperm motility in semen by depressing the pHi. A simple hypothesis that explains these observations is that the volatile motility inhibitor is COs, which could acidify pHi as a diffusable weak acid. In this regard, sperm diluted into seawater release acid, and this acid release is related to the pHi increase and motility initiation. In fact, nearly half of the acid released by sperm upon dilution is volatile and may therefore be due to COz efflux. Most of the acid, however, cannot be attributed to COs release because it is not volatile. Thus, when sperm are diluted into seawater, they raise their pHi by releasing COP and protons from the cytoplasm into the surrounding seawater.

correlation between pHi increase and motility initiation in S. purpuratus sperm. Also consistent with the idea Sea urchin spermatozoa are immotile in semen and that pHi might be a regulator of sea urchin sperm mobecome intensely motile after dilution into seawater. tility is the report of Goldstein (1979) that dememRecent studies indicate that a Na+-dependent increase branated L J&%T and S. purpuratus spermatozoa cannot in intracellular pH (pHi) may be the trigger for this be reactivated to move at pH’s of 7.3-7.5, swim feebly motility initiation. Nishioka and Cross (1978) showed at pH 7.5-7.7, and have maximum motility at that acid is released from sea urchin spermatozoa (Ly- pH 8.0. tech&us p&us and Strongylocentrotus purpuratus) upon These studies, however, do not explain why sperdilution of semen into seawater. They found that diluting matozoa are immotile in semen. High levels of Na+ are sperm into Na-free seawater (ONaSW) prevented acid present in the semen of many species of animals (e.g., release and motility initiation, while the subsequent Mann and Lutwak-Mann, 1981), including sea urchins addition of Na+ (10 mM) initiated motility and acid (as we show in this paper); therefore, if a Na-dependent release; also, ammonia addition, which might be ex- alkalinization initiates motility upon dilution of sea urpected to raise pHi, also initiated motility in the absence chin sperm, why shouldn’t this occur in semen? Two of Na+. These results provided the initial indication that possibilities seem likely: either a motility inhibitor is a Na-dependent increase in pHi was probably the trigger present in semen, or else a metabolic requirement (e.g., for sperm motility initiation. Lee et al (1980,1983) used 0,) is lacking. With regard to inhibitors, Cohn (1918) molecular probes to directly monitor changes in pHi and presented evidence that carbon dioxide produced by found that a pHi increase of 0.4-0.5 unit took place when sperm in concentrated cell suspensions can paralyze motility was initiated by adding Na+ or NH: to L. pictus spermatozoan movement. Because semen is a dense sussperm in ONaSW. Christen et al. (1982) showed a similar pension of cells, dilution might therefore permit motility by decreasing the CO2 tension. In agreement with this, Runnstrom et al. (1944) noted that even in concentrated 1 Present address: The Biological Laboratories, 16 Divinity Avenue, semen, motile sperm can be observed transiently near Harvard University, Cambridge,Mass. 02138. the edges of the semen drop; they suggested that COZ ‘To whom reprint requests should be addressed. INTRODUCTION

493 6012-1606133 $3.00 Copyright All rights

0 1983 by Academic Press. Inc. of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

is inhibitory and diffuses away at the semen-air interface, permitting sperm activity. These results are also consistent with the observations of Mohri and Yasumasu (1963), who found that COz reversibly inhibits the respiration and motility of sea urchin sperm. Other workers, however, have proposed that high concentrations of potassium or hydrogen ions, proteins, or other inhibitory substances in the seminal fluid are responsible for the immobility of sperm in semen (see Rothschild, 1948). Rothschild (1948) examined many of these ideas in detail. He found that the potassium concentration in seminal fluid of the sea urchin Echinus was about 20 milf (as opposed to 10 mM in seawater) and the pH was 7.3-7.7 (the pH of seawater is 7.9-8.2). Sperm diluted into seawater containing 20 mlMK+ were motile, as were sperm in seawater considerably more acidic than pH 7.3. Rothschild (1948) and Hayashi (1945) also found that if a few sperm were diluted into seminal fluid, motility was initiated. In addition, sperm in undiluted semen could be made motile by increasing the O2tension in the atmosphere surrounding the semen. On the basis of these experiments, Rothschild reasoned that sperm were not rendered immotile by the pH, [K+], or any other nonvolatile compound in semen. Furthermore, flowing nitrogen gas over semen did not activate the sperm, and the oxygen content of semen was determined to be 10% that of air. He therefore concluded that Oz deprivation was most likely responsible for the lack of movement in semen (Rothschild, 1948). The present study reinvestigates the problem of why sperm are not motile in semen. Extending the previous studies which implicate pHi increases as regulators of sperm motility, we used 31P-NMR to measure the pHi of sperm in semen. Nonmotile sperm in undiluted semen were found to have a pHi which is 0.36 pH unit lower than that of motile sperm which have been diluted into seawater. Dilution, however, is not necessary to initiate motility, since gassing thin layers of semen with air or even Nz can initiate motility. This suggests that a volatile substance (possibly COz) inhibits sperm motility in semen. Even though the inhibitor in semen is volatile, only 43% of the acid released by sperm upon dilution into seawater is volatile. Consequently, sperm release 57% of their acid as protons, not as COZ. With regard to the nonvolatile acid, we suggest that the volatile inhibitor (e.g., COZ) lowers pHi either (1) by inhibiting the pHi regulatory system of the sperm or (2) by entering the sperm as a neutral weak acid, but then leaving as a negatively charged ion (e.g., HC03), thus leaving behind H+ ions. Upon dilution of semen, COz diffuses into the surrounding seawater and protons are transported from

VOLUME 98, 1983

the sperm cytoplasm, thereby producing the observed pHi increase and motility. MATERIALS

AND

METHODS

Handling of Sperm Except for experiments on the ionic content of semen, the semen of Lytechinus picks and Strongylocentrotus purpuratus was collected by injecting males of these two species with 0.2 or 5 ml, respectively, of 0.5 M KCl. The semen was stored on ice in microcentrifuge tubes and all experiments were conducted within 6 hr of semen collection. Seawater Fomzulations Artificial seawater (ASW) had the following composition: 493 mM NaCl, 10 mlM KCl, 10 mM CaClz, 27 mM MgClz, 28 mM MgSO*, 2.5 mAf NaHC03, and 10 mM Hepes (pH 8.0). For Ca2+-free seawater (OCaSW), the CaC12was deleted. Na+-free seawater (ONaSW) was prepared fresh daily by substituting choline Cl for NaCl and KHC03 for NaHC03 in the ASW formula. COP-free ASW was prepared by deleting both NaHC03 and Hepes from ASW, adding 1 mM glycyglycine, and bubbling humidified N2 gas through the solution overnight before use. “P-NMR Measurement of PHi Intracellular pH of L pictus sperm cells was measured by determining the chemical shift of the inorganic phosphate (Pi) resonance peak at 81 MHz in a Nicolet Magnetics NT-200 NMR system. This method of pH1 determination depends on the observation that the position of the Pi resonance peak in the phosphorus NMR spectrum is a function of the pH of the phosphate’s environment. The Pi standard curve (Pi chemical shift versus pH) and conditions of spectra measurement are described by Winkler et aL (1982). Semen from 40 males was collected, and sample sizes of 4 ml (for undiluted semen) or 7 ml (for semen diluted 1:6 or 1:lO) were used to obtain the three spectra reported (Fig. 1 and Table 1). For undiluted semen, the chemical shift in ppm was determined in an unaerated sample. This spectrum (Fig. 1A) required less than 1 minute to obtain. To activate motility, semen was diluted 1:lO with OCaSW (Fig. lB), then the new chemical shift of Pi was measured. [OCaSW was used as the dilutent to maximize the motile lifetime of the sperm (see Lee et al, 19&3).]Because of the dilution, a minimum of 4 min was required to obtain a clear spectrum. These diluted samples were also measured without aeration, because the high concentration of cells and protein caused the sample to bubble over if aeration

JOHNSON

ET AL.

Inhibition

was attempted; therefore, these samples may have become anaerobic. To minimize this problem, the results reported here come from only those spectra which exhibited large creatine phosphate peaks. Also, sperm samples were withdrawn concurrently to assay motility. Atomic Abwrption Spectrophotometrg The ionic composition of L. pi&us seminal fluid was determined by atomic absorption spectrophotometry. For these experiments, seminal fluid was collected and prepared in different ways to minimize ionic contamination of the seminal fluid by seawater on the outside of the urchin or by the KC1 used to induce spawning. First, the male urchin was washed carefully with 0.5 M choline chloride, then blotted dry and spawned. This procedure yielded semen that had less than 0.5% of its volume contributed by the seawater originally coating the urchin (as determined with tracer amounts of [3H]sorbitol in the seawater used to wash the urchin). Second, the ionic composition of seminal fluid collected by injection of KC1 or by injection of about 2 ml of air per urchin was compared. If injecting KC1 increases the [K+] of seminal fluid, then the K+ concentration of seminal fluid collected by injecting air should be less. After concentrated semen had been collected, it was spun for 1 min (at 12,000~;4°C) in an Eppendorf microcentrifuge 5412 (Brinkmann Instruments, Inc., Westbury, N. Y.) and the supernatant was transferred to another tube. This supernate was successively spun and transferred three more times to remove any residual sperm and then used for the ionic and protein measurements of seminal fluid. Seminal fluid prepared by this procedure from concentrated semen constituted less than 20% of the total volume of the original semen sample. The concentration of Na, K, Ca, and Mg in these seminal fluid samples was then assayed with a Perkin-Elmer Model 303 atomic absorption spectrophotometer. Standard assay procedures were used as described in the “Manual of Methods for Chemical Analysis of Water and Wastes” (1974). Gas Chamber Semen (which had been stored on ice for about 2 hr) was spread as a thin film on a washed glass slide in a humidified 30-ml Falcon tissue culture flask. This arrangement allowed visual inspection of the sperm through a low-power microscope (4X objective with a 10X ocular). The flow-through chamber was sealed except for inlet and outlet hoses. The outlet hose was placed in a beaker of water so that no back-diffusion of air could occur when gas flow was stopped, and the inlet hose was connected to gas tanks via a bubbler which

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saturated the gas with water at 18°C. Gas flow rates of 50-100 ml/min were used. Nitrogen, oxygen, and carbon dioxide gases were 99.996% pure, and air was obtained from a laboratory compressed air jet. The microscopic observations of sperm in semen were recorded on videotape. Later, the motility was scored by three indifferent observers. For experiments with diluted sperm, motility was assessed as percentage motile sperm. For observations on undiluted semen, the sperm are too concentrated to determine percentage motility, so a subjective grading system was used: 0 = no moving sperm, 1 = barely detectable motility, 2 = intermediate motility, and 3 = high motility. The flow of gas over semen did not create artifactual sperm movement. RESULTS

“P-NMR Measurement of Sperm pHi Intracellular pH has been implicated as the regulator of motility initiation in sea urchin spermatozoa (Nishioka and Cross, 1978; Lee et a& 1983; Christen et aL, 1982; Goldstein, 1979). Some of these studies were performed on sperm under nonecological conditionsnamely, semen diluted into Na-free ASW. We wanted to monitor the pHi of sea urchin sperm before and after dilution of semen into Na-containing seawater to see if the same alkalinization would be observed. Molecular probes such as those used by Lee et al. (1983) cannot be used for assaying the pHi of sperm in semen because the cell suspension is too concentrated. Therefore, we have here used 31P-NMR, which is ideal for such conditions. Figure 1A depicts the 31P-NMR spectrum of undiluted semen; peak b is the inorganic phosphate peak, which is sensitive to pH. As shown in Table 1, the position of the inorganic phosphate peak (2.620 ppm) on the phosphorus spectrum indicates a pHi of 7.24, as determined from a titration curve using media mimicking the ionic environment of L pi&xs egg cytoplasm (Winkler et aL, 1982). When semen (external pH of 7.1) was diluted sixfold with OCaSW buffered to pH 8, there was little activation of motility (motility level 1) and little or no pHi change as seen by examining the phosphate peak (Table 1). [OCaSW was used to prevent a gradual reacidification which occurs following the dilution of sperm into seawater; this reacidification results from a Ca2+uptake (Lee et aL, 1983). As the NMR reading requires several minutes, this reacidification could have masked any earlier pHi changes.] This indicates that the inorganic phosphate is intracellular, because if it were external, the position of the phosphate peak would have been shifted upon exposure to media buffered at pH 8.0. Prominent organic phosphates are

496

DEVELOPMENTAL BIOLOGY

VOLUME 98, 1983

b

A a

a

B

2b

IO

0

-10

-20

PPM

FIG. 1. *lP-NMR spectrum of L pictwa spermatozoa at 19°C. (A) Spectrum of undiluted semen containing nonmotile spermatozoa. (B) Spectrum of semen diluted 1:lO in OCaSW and containing motile spermatozoa. The peaks are (a) a standard of methylenediphosphonic acid encased in a capillary; (b) inorganic phosphate; (c) creatine phosphate; and the phosphates of ATP-7 (d), (Y (e), and @ (f).

at c (creatine phosphate) and d, e, and f (y-, (Y-, and /3phosphates of ATP). When sperm are diluted lo-fold into OCaSW, motility is activated (motility level 2.5) and the pHi as measured

by the inorganic phosphate peak rises to 7.60 (Fig. 1B and Table 1). This measurement required about 4 min to obtain, and although the background is noisier in this spectrum (Fig. 1B) than that obtained with undi-

JOHNSON ET AL.

TABLE 1 31P-NMR DETERMINATIONOFpHi IN L. pi&s

Semen sample Undiluted semen Semen diluted 6X Semen diluted 10X

Motility level 0 1 2.5

Inhibition

SPERMATOZOA

Chemical shift of Pi (ppm)

pHi

2.620 2.586 2.876

7.24 7.19 7.60

Note. Undiluted semen and semen diluted either 6X or 10X in OCaSW were assayed.

luted semen (Fig. lA), the Pi peak is still sharp and readily identifiable. In subsequent measurements of the same sample, pHi progressively fell back to about 7.2 (by 14 min) and motility ceased. Therefore, the activation of spermatozoan motility is accompanied by an alkalinization of at least 0.36 pH unit. This may be a low estimate since the high cell concentrations demanded by the NMR technique (see Materials and Methods) did not allow as robust an activation of motility as is typical of more dilute suspensions (e.g., 1:lOO or more). The 31P-NMR data also provided information on energy levels in Lytechinus spermatozoa before and after motility initiation. Comparisons of NMR measurements on undiluted sperm and sequential NMR measurements of sperm diluted lo-fold into OCaSW showed that creatine phosphate levels remained high in the early part of the experiment (4-8 min after dilution), but ATP levels had decreased dramatically by this time. This decrease correlates well with ATP measurements by other methods which show that ATP levels drop after motility is initiated upon dilution of L pi&us (Johnson, 1981) and Hemicentrotus pukhewimus (Hino et al, 1980) sperm. In addition, these results confirm that low levels of ATP are not responsible for the lack of sperm motility in semen.

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Artificial seawater of the composition of the seminal fluid (as listed in Table 1) will fully support motility. Therefore, hypotheses that elevated K+, reduced Na+, or changes in Mgz+ or Ca2’ are responsible for spermatozoa immobility in semen would not appear to be valid. Both Hayashi (1945) and Rothschild (1948) reached this same conclusion.

Initiation

of Motility in Undiluted Semen

Sperm in a film of undiluted semen in a dry atmosphere are not observed to move, except transiently near the edges of the film. If, however, a film of undiluted semen is placed in a humidified chamber, the sperm gradually become motile throughout most of the film and can attain high levels of movement (Fig. 2A). This suggests that gas exchange at the edges permits motility initiation, but that in a dry atmosphere the edges desiccate rapidly, killing the motile sperm. In support of this hypothesis, the process of motility initiation in undiluted semen under humid conditions can be accelerated and accentuated if water-saturated air is continuously flowed over the semen. Humidified oxygen works even better than air (data not shown). These experiments show that sperm can be motile in undiluted semen if suitable conditions are met (as in a thin film in a humid environment). These data also indicate that sperm are not immotile because of the ionic composition, protein content, or presence of nonvolatile inhibitors in seminal fluid. Therefore, a volatile substance seems to be responsible for lack of motility. It could be that increasing the gas exchange of semen is removing a volatile inhibitor or is boosting the oxygen tension in previously anaerobic semen. However, if water-saturated nitrogen instead of air is flowed over the semen film, it will also activate motility, albeit for a TABLE 2

Ionic Composition of Seminal Fluid Table 2 lists the composition of L. pi&us seminal fluid (collected by KC1 or air injection) as compared with ASW; the ionic composition found is similar to that described by Rothschild (1948) for the seminal fluid of Echinus esculentus. It is also the same as the composition of Strongylocentrotus purpuratus seminal fluid as measured by atomic absorption spectrophotometry and Xray microanalysis (R. Schackmann and M. Cantino, personal communication), except that the values for potassium were slightly higher (26 mM) in S. purpuratus seminal fluid. (Seminal fluid was obtained from S. purpuratus by injecting KCl, which may have increased the potassium content slightly, as it did to L. pi&us seminal fluid-Table 2.)

COMPOSITION OF L. pictus SEMINAL FLUID

Na K Ca Mg Protein” pHd

Seminal fluid”

Seminal fluidb (mkf)

ASW bW

440 mM 19 mM 9.7 mM 48 mM 0.58 mg/ml 7.5-7.6

520 23 9.7 46

495 10 10 55 8.0

7.5-7.6

a Collected by injecting urchins with air (see Materials and Methods). bCollected by injecting urchins with minimal amounts of KC1 (see Materials and Methods). ’ Determined by the Bio-Rad protein assay. d Determined with a glass pH electrode. (pH of undiluted semen is about 7.1).

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o+ I , , 0

2

4 minutes

6

8

2 4 minutes

0

6

FIG. 2. (A) Activation of L pi&us sperm motility in undiluted semen in a humidified chamber. Motility was scored as described under Materials and Methods section by three impartial observers watching a video replay of the experiment. For any given time, each point is the motility assessment given by one of the scorers. The line connects the average of each triplicate. (B) Activation of L picks sperm motility in the humidified chamber as in A, except gases were flowed over the semen. The bars above the figure refer to the times when nitrogen or air, respectively, were flowed through the chamber.

briefer time (Fig. 2B). This result therefore suggests that boosting O2 tension is not the factor, but that removal of a volatile inhibitor is a sufficient condition for allowing sperm motility in semen. COz is a good candidate for this inhibitor, since it quickly and reversibly immobilizes sperm (Fig. 3). In Fig. 3A, sperm in undiluted semen are activated with air; then 5% CO2in air (calibrated with gas flowmeters) is passed over the semen. Within 0.5 min, the high level of motility is eliminated. If the COz-air flow is stopped and the chamber sealed, motility never reappears. Figure 3B illustrates the reversibility of COz inhibition. If air without added COz is passed over COz-inhibited semen, motility reappears within 2 min and persists. Even N2 will now reinitiate long-term motility, as depicted in Fig. 3C. [The longer duration motility here (Fig. 3C), as opposed to that seen in Fig. 2B, probably results from the prior oxygenation of the semen used for the experiment in 3C.l The experiments depicted in Fig. 3 were done with undiluted semen, but similar results were obtained if the semen was diluted 1:l or 1:2 with seawater. Also, we observed that Oz, air, or N2 cannot activate motility of sperm suspended in ONaSW, so unlike semen immobilization, ONaSW immobilization cannot be released by increased gas exchange. Similar results to those shown in Figs. 2 and 3 were also obtained with S. purpuratus semen. The only significant difference was that S. purpuratus sperm were able to recover from the hypoxia produced by flowing Nz gas over semen. That is, in contrast to the results with L pictm shown in Fig. 2B, nitrogen-treated S. mrpuratus sperm regained motility when air was flowed over them. To summarize these results, sperm motility in un-

diluted semen (of either L. pi&us or S. purpuratus) can be initiated by increasing the gas tlow over the semen. Activation is not caused simply by providing Oe, since Nz was also effective. COz inhibited motility, and this inhibition could be reversed by removing the COz with air or Nz flow. These results are most simply interpreted as showing the presence of a volatile inhibitor of motility in semen. As COz inhibits motility and also is volatile

0

2

4

6

8

IO

12

14

MINUTES FIG. 3. Carbon dioxide inhibition of motility in L pictus semen and its reversibility by air or nitrogen. Bars above figure indicate when gases were passed over semen. In A, semen was not otherwise treated. In the other two graphs, the CO* inhibition of motility was reversed by subsequent flow of air (B) or nitrogen (C) over the semen.

JOHNSON

ET

AL.

Inhibition

at pH 7, it is a reasonable candidate for this motility inhibitor. Acid Released by Sperwt is Nonvolatile

A simple hypothesis to explain how sperm motility is initiated upon dilution (or gas flow) is that a volatile motility inhibitor maintains a low sperm pHi; upon dilution, this substance falls below an effective level and the pHi increases. This hypothesis is supported by our NMR data which show that the sperm pHi increases 0.36 pH unit upon dilution. This pHi change is also similar in magnitude to the Na+-dependent pHi increase associated with motility initiation when Naf is added to sperm previously diluted into Na+-free ASW (Lee et al, 1983). Since COe reversibly inhibits sperm motility (Fig. 3) and is also known to acidify cells by acting as a diffusable weak acid (e.g., Roos and Boron, 1981), we wondered whether the acid released by sperm upon dilution into seawater (Nishioka and Cross, 1978) might be volatile as well. We used a method similar to that of Gillies et al. (1981) and Holland and Gould-Somero (1982) in which CO2 is driven off by bubbling N2 gas through the suspension (in the presence of carbonic anhydrase) and monitoring CO2efflux by the resultant change in external pH. For these experiments, semen was diluted into COzfree ASW; and to minimize COaproduction by metabolic activity of the sperm, antimycin (a mitochondrial blocking agent) was added. When the pH had stabilized following dilution of semen into the COz-free ASW (a in Fig. 4A), the sperm were removed by centrifugation. Then, while the pH of the supernatant was monitored, bubbling with Nz gas was resumed, and carbonic anhydrase was added to accelerate the conversion of HCO, to COz. The results (Fig. 4A) show that 57% of the acid released was nonvolatile: when sperm were diluted into seawater, the extracellular pH dropped from 7.85 (H+ = 1.4 X lo-* M) to 7.55 (H+ = 2.8 X 10m8M), and Nz bubbling restored the seawater pH to 7.65 (H+ = 2.2 X lop8 M). Therefore, of the total acid released by sperm (1.4 X 10m8M), 43% was volatile (0.6 X 10m8M). Figure 4B shows that COz gas produces a pH change that is volatile and totally reversible upon aerating the solution with Nz gas. (Also note from Fig. 4B that two equal, sequential additions of HCl lower the pH by about the same amounts as do the volatile and nonvolatile components of the acid released by sperm in Fig. 4A. This observation reinforces the above calculations demonstrating that the volatile and nonvolatile components of the acid release each contribute approximately 50% to the total acid released.) Seminal plasma does not

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produce the observed pH changes (Fig. 4C). Therefore, 57% of the intracellular alkalinization results from release of protons or a nonvolatile weak acid from spermatozoa and not by passive diffusion of COa from the cytoplasm. DISCUSSION

Our results show that sperm motility in semen is limited by a volatile substance (since motility can be initiated if gas exchange is promoted under conditions that maintain high humidity) and the volatile substance is not a missing metabolic requirement such as O2(since gassing with Na initiates motility); therefore, the volatile substance must be an inhibitor. We have also shown that the pHi of sperm in semen is lower than the pHi of diluted sperm (NMR data) and that 57% of the acid released by the sperm during this alkalinization is not volatile. These results agree in part with those of previous workers who concluded that a volatile substance was responsible for the lack of sperm motility in semen (e.g., Cohn, 1918; Runnstrom et a& 1944; Rothschild, 1948; Mohri and Yasumasu, 1963). However, our results are the first to show clearly that the volatile substance is an inhibitor. Rothschild concluded that O2was limiting because he could not activate motility by blowing Nz gas over semen; however, he was apparently using dry gases and the sperm may have been damaged by desiccation. Using humidified N2 gas, we were able to initiate motility; therefore Oz is not limiting and motility must be inhibited by a volatile substance in semen. How does this volatile substance inhibit sperm motility in semen? Two possibilities seem likely; either the volatile inhibitor in semen acts directly on the motility apparatus of the sperm (and the observed pHi changes are a coincidence) or else the volatile inhibitor acts by controlling the sperm pHi. A direct effect of COZ,perhaps by carbamylation of proteins, is suggested by the results of Brokaw (Brokaw and Simonick, 1976; Brokaw, 1977), who found that CO2 directly inhibited the beating of demembranated sea urchin spermatozoa. However, this COz inhibition only occurred above pH 8.0, whereas the range of measured pHi values for immotile and motile spermatozoa are 6.3-7.2 and 6.7-7.9, respectively (Schackmann et a& 1981; Christen et al, 1982; Lee et aL, 1983; our NMR results). Therefore it seems unlikely that COz acts directly on the motile apparatus at physiological pH’s. The alternative explanation-that the volatile inhibitor acts by controlling pHi-appears to be more likely since (1) a pHi increase (of 0.4-0.5 pH unit) is a prerequisite for motility initiation of sperm diluted into

DEVELOPMENTALBIOLOGY ASW Ant I v Sperm li

B

C

FIG. 4. Dilution of S. pwpuratus semen into COs-free ASW produces a nonvolatile acidification. Two milliliters of CO*-free ASW (see Materials and Methods) was placed in a temperature-controlled (19°C) chamber through which humidified Ns gas was continuously bubbled. The pH was continuously monitored, and at the arrow marked Ant, the mitochondrial blocking agent, antimycin (5 PM) was added. (A) 50 pl of semen was added (at the arrow marked sperm), and after the pH had stabilized (at a), the semen plus ASW mixture was removed, centrifuged (12,OOOg,1 min, 4’C), and the sperm-free supernatant was returned to the chamber at b. Carbonic anhydrase (50 pg/ml) was added (at CA) and the pH was monitored for 13 min. Extrapolation of this curve to its asymptote indicated the pH would stabilize at 7.65 + 0.01. Finally, 10 ~1 of 0.01 N HCI was added (at HCI) to determine the buffering capacity of the solution. This experiment was repeated three times with two semen samples, and all produced similar results (final pH ranged from ‘7.65 to 7.68). Between pH 7.5 and 8.0, less than 2% of S. purpuratus spermatozoa undergo acrosome reaction in the absence of egg jelly (as found also by Collins and Epel (19’77) and Schackmann et al (1978)) and therefore acrosome reactions do not contribute significantly to the acid release depicted in this figure. (B) CO2 control. COz gas (100%) was briefly flowed over CO*-free ASW (plus antimycin) at the arrow marked COa. Then the protocol used in A was repeated: the solution was removed and centrifuged (at a), replaced at b, carbonic anhydrase was added (at CA), and the pH was monitored. Extrapolation of this curve to its asymptote indicated the pH would stabilize at 7.85 ? 0.015. The pH change produced by each of the lo-r1 aliquots of 0.01 N HCl was similar to that produced by the HCl added in A; therefore, there was no great difference in the buffering capacity in the two experiments. (C) Seminal plasma control.

VOLUME98.1983

ONaSW (Lee et aL, 1983; Christen et ah, 1982), (2) a pHi increase of similar magnitude occurs upon dilution of sperm into seawater (Table l), and (3) demembranated sea urchin sperm have a high pH sensitivity for motility, being quiescent at pH 7.3-7.5 but highly active at pH 8.0 (Goldstein, 1979). It is therefore also probable that when semen is gassed with Nz or 02, the motility initiation is also accompanied by an increase in pHi and that this is a prerequisite for motility initiation. What is the mechanism by which the inhibitor (e.g., COZ) depresses pHi of sperm in semen? One possibility is that COa diffuses into the sperm and thus depresses the pHi by a weak acid effect (e.g., McLaughlin and Dilger, 1980; Roos and Boron, 1981). However, since 57% of the acid released upon dilution of semen into seawater is not volatile (Fig. 4), a maximum of 43% of the acid release could result from the efflux of COz or some other volatile substance. The other 57% of the acid must ensue from release of a nonvolatile acid. It seems likely that two mechanisms could account for nonvolatile acid accumulation by sperm in semen. In one mechanism, a volatile substance (such as CO,) could still acidify the sperm by a weak acid effect, but only if the charged form of the acid (e.g., HCO,) were transported out of the cell, thus leaving behind protons. Since by this mechanism, the weak acid could repeatedly shuttle into and out of the sperm cells (effectively carrying H+ in or OH- out), a low concentration of weak acid could produce the depressed pHi of sperm in semen. An alternative is that the volatile motility inhibitor (COJ controls sperm pHi by interacting with the Na+dependent membrane pumps which regulate sperm pHi. Either of these mechanisms would produce a net cytoplasmic accumulation of protons, which are then transported out of the sperm cell by a Na+-dependent mechanism upon dilution of semen into seawater. The chemical nature of this volatile sperm motility inhibitor is unknown, although COa is an attractive candidate since it is present in semen. The level of CO2 in semen could be controlled by a simple feedback regulation of sperm metabolism: COB inhibits metabolism by lowering the pHi, and whenever the COz level decreases below the inhibitory threshold, metabolism turns on, producing more CO, which turns metabolism off again-an idea which harkens back to models of morphogenesis proposed by Loomis (1961). In conclusion, we propose the following model for regOne milliliter of semen was centrifuged (12,OOOg,4”C, 2 min) and 50 ~1 of the seminal plasma was added to COr-free ASW (at SP); carbonic anhydrase was added to CA. This shows that nearly all of the nonvolatile acidification observed when semen is added to ASW (as in A) is from the spermatozoa and not from the seminal plasma.

JOHNSON ET AL.

Inhibition

ulation of sperm motility in semen and upon dilution into seawater. In semen, a volatile inhibitor (possibly COP) inhibits sperm motility by depressing the sperm pHi. Upon dilution of sperm into seawater, volatile and nonvolatile acid efflux allows pHi to rise sufficiently to initiate sperm motility. We wish to acknowledge the Nuclear Magnetic Resonance Facility (at the University of California at Davis) for the use of the Nicolet200 NMR spectrometer and the assistance of Dr. Gerald B. Matson in operating the spectrometer. Also, we thank Drs. R. Schackmann and M. Cantino for sharing their seminal plasma data, and Drs. L. F. Jaffe and H. Mohri for assisting in documenting the historical development of these ideas. M. M. Winkler is a fellow of the Jane Coffin Childs Memorial Fund for Medical Research, and the remainder of this work was supported by a grant from the National Science Foundation.

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