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Chapter 4 Ion Measurements in Sea Urchin Sperm
Chapter 4 Ion Measzlrements in Sea Urchin Sperm ROBERT SCHACKMANN Department of Biorbemistiy University of Warbington Seattle, Warbington
1. 11.
HI. IV . V. VI.
............
............
A. Sperm Preparation .......................... B. Measurement of lntracellular and Extracellular Water Space . . . . . . . . . . . . . . . pH Measurements Important to Sperm Activation ............................ A. Measurement of Acid Efflux ......................................... B. Measurement of pHi ................ Measurement of Cation Up A. Measurement of Ca2+ ....................... B. Measurement of Na+ and K + ................ . . . . . . . . . . . . . . . . . . . . . . . Measurement of Membrane .............. Conclusion . . . . . . . . . . . . . .............. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
57 58 58
59 60 60 61
66 68 69 70
Introduction
Recent literature, both biochemical and physiological, pays increasing attention to relationships among cellular ion movements, ion-translocating systems, and cellular function. Ion transport, once thought to be specific for tissues containing excitable cells or cells specialized for salt or acid secretion, appears now to be a component of cell responses to stimuli throughout growth and development. Na+ / H + exchanges, which increase pHil (Johnson et al., 1976; Shen and Steinhardt, 1978; Hesketh et al., 1985; L'Allemain et al., 1984; Moolenaar er al., 1984; Rothenberg er al., 1983), changes in [Ca2+Ii(Ridgway 'Abbreviations: pHi, intracellular pH; pH,, extracellular pH; [Ca* I,, intracellular calcium concentration; Ar, accumulation ratio; TPP, tetraphenylphosphonium; SCN, thiocyanate; DMO, 5.5dimethyloazolidine-Z,4-dione;P,, inorganic phosphate. +
57 METHODS IN CELL BIOLOGY. VOL. 27
Copyright 0 1986 by Academic R s s . Inc. All rights of reproduction in any form reserved.
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ROBERT SCHACKMANN
et al., 1977; Steinhardt er al., 1977; Hesketh et al., 1985), or changes in the plasma membrane potential (Rothenberg et al., 1982) are among the earliest responses of quiescent eukaryotic cells to stimulation of cell growth. Cellular responses to a variety of hormonal agonists have been linked to both alterations of [Ca2+Ii (Thomas et af., 1984) and pHi (Moore et al., 1982), and ATP production by oxidative phosphorylation (Boyer et al., 1977) involves pH and potential gradients operating at the subcellular level. These are but a few examples of biological systems in which ions serve as regulatory components of cell biology. Historically, echinoid sperm were among the first cells used to provide evidence that ionic changes are important to cell activation. It was demonstrated 3C years ago by Jean Dan that the sea urchin sperm underwent an acrosomal reaction when exposed to factors released from the egg (egg jelly). This reaction was defined morphologically to consist of exocytosis of the acrosomal granule and polymerization of actin into the acrosomal rod. Dan (1954) showed that removal of Ca2 from the seawater prevented the acrosomal reaction. Likewise increasing the pH, to 9.2 spontaneously initiated the acrosomal reaction in the absence of any of the egg factors (Dan, 1952). Thus, ionic alterations were implicated in the activation of the sperm for fertilization. We now know that egg factors that initiate the acrosomal reaction cause both Ca2+ influx and increased pHi (Schackmann et al., 1978, 1981). The Ca2+ influx is thought to lead to exocytosis of the acrosomal granule while increased pH, can cause actin polymerization (Tilney et al., 1978; Schroeder and Christen, 1982). Additionally, pH, is a primary regulatory component of sea urchin sperm motility and respiration (Christen et al., 1982, 1983a,b; Lee et al., 1983). At low pH, (< -7.0) sperm are immotile. As pH, is increased to the normal pHi of -7.4, sperm respiration and motility are initiated. These findings underlie the importance for studying ion movements as regulatory components of sperm activation. This article discusses methods used to measure ion changes important to activation of sea urchin sperm motility and the acrosome reaction. I describe briefly the methods used by several laboratories to measure pH,, ion fluxes, and membrane potentials in sea urchin sperm. It is not my intention to explain in complete detail how to make the ion measurements, but to provide a supplement to published methods which hopefully contains additional insight on their application. +
11. A.
Sperm Preparation and Determination of Intracellular Water Space
Sperm Preparation
Sperm are typically collected from sea urchins by injecting a few milliliters of 0.5 M KCl into the coelomic cavity. After initiation of spawning, the animals are
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
59
inverted and allowed to shed into a Petri dish without seawater (“dry sperm”). Experiments are usually performed at 0.1 to 10% suspensions of “dry sperm” (vo1ume:volume). The supernatant of centrifuged dry sperm has been analyzed by atomic absorption spectroscopy (Johnson et al., 1983) and except for [ K + ] (20-30 mM), the composition approximates that of seawater. Thus “a+] is > 400 mM and [Ca2+] is -10 mM. The high “a+] necessitates that sperm be washed, if they are to be removed from Na+ . A 100-fold dilution contains -4 mM Na . This is sufficient Na to activate sperm motility and respiration (Christen et al., 1982). Washing of sperm is easily accomplished by gentle centrifugation at IOOO-1500 g for -10 minutes. +
+
B . Measurements of Intracellular and Extracellular Water Space It is convenient to measure uptake and cell content of ions, expressed per unit of cell protein or cell number. However, interpretation of the physiological significance of the ion content often requires an approximation of the intracellular concentration, which requires evaluation of the intracellular water space. The accepted method for evaluating intracellular water consists of a difference measurement between total [3H]H20 in a pellet of cells and the pellet volume occupied by molecules impermeant to the plasma membrane (Rottenberg, 1979). Water rapidly penetrates biological membranes and the volume of [3H]H,0 in a pellet represents the total of intra- and extracellular water space. Typically, sugar molecules fill the role as impermeant, extracellular markers. [ ‘‘C]Inulin, sucrose, and sorbitol have all been used with sea urchin sperm (Schackmann et al., 1981; Lee et al., 1983). Values for the extracellular space are -0.2 pl/IO* sperm. The intracellular water space is 0.7-0.8 pl/ lo* sperm (Schackmann et al., 198 I ; Lee et ul., 1983) and is quite close to the morphologically determined value of 0.72 p1/ lo* sperm for the sea urchin A. punctulatu (Harvey and Anderson, 1943). Sucrose is easier to use than carboxy-inulin as the latter tends to accumulate precipitative radioactive material which can lead to erroneously large volumes for extracellular space (in extreme cases exceeding the total [”H]H2O space). However, sucrose is finitely permeant and incubations as an extracellular marker should be kept to only a few minutes. Since the intracellular H 2 0 space measurement is determined by a difference between experimental values, each with its own inherent experimental error, the total error in the intracellular water content can be larger than that for measurement of uptake of the ions or pHi probes themselves. Small differences or changes in intracellular volume are also not easily detected. It is clearly important to evaluate the cell and pellet water spaces under each set of experimental conditions used. The accuracy of the calculations of ion concentrations, pH,, or membrane potential are no better than the water space measurements themselves.
60
ROBERT SCHACKMANN
111. pH Measurements Important to Sperm Activation Initiation of motility and induction of the acrosome reaction are associated with increases in pH, regardless of the stimulus applied. To be motile the sperm pH, must be 2 -7.2 (Christen et af., 1982, 1983a,b; Lee et af., 1983). For the acrosome reaction to occur the pHi must be raised to -7.6 (Schackmann et af., 1981). However, the experiments first indicating the importance of pHi increases in sperm activation were not based on direct measurements of pHi, but rather were observations that acid efflux occurred upon activation of either motility or the acrosome reaction. Activation of sperm respiration and motility by dilution (Johnson et af., 1983), by addition of the egg peptide “speract” (Hansbrough and Garbers, 1981; Repaske and Garbers, 1983), or by addition of Na+ to sperm in Na+-depleted seawater (Nishioka and Cross, 1978; Lee, 1984a), all result in acid efflux and elevation of pHi. Induction of the acrosome reaction by either egg jelly or ionophores was likewise found to be accompanied by acid efflux (Schackmann et af., 1978; Tilney et af., 1978).
A.
Measurement of Acid Efflux
Measurement of the acid efflux upon activation of motility or the acrosome reaction provides rapid quantitative information necessary to understand how H + efflux is coupled to other ion movements such as Na+ entry (see Section IV,B). To measure the efflux a pH electrode is placed in a sperm suspension, agents are added to stimulate the sperm, and changes in the external pH are followed with a recorder. For example, when Na+ is added to a 1% sperm suspension in Na+-free seawater, H + efflux of -100 nmol occurs. The resulting pH, decrease is easily monitored with most of the better modem pH meters and electrodes. Our laboratory uses a pHM64 Radiometer pH meter with a GK2310-C combination electrode. The output to the recorder must be offset (Madeira, 1975). With seawater buffered with HC0,- (Lee, 1984a) or low concentrations of Tris or HEPES (- 1 mM), variation of the sperm concentration allows manipulation of the total external pH change. I try to keep the change to -0.05 pH units to approximate measurements at a constant pH,. The response time of the electrode must be reasonably fast as the rapid pH changes due to activation of the acrosome reaction and motility initiation are complete within 15 seconds. Calculation of acid equivalents released is usually performed by titration with freshly prepared NaOH or KOH. Stock solutions should be standardized against an acid of known concentration since hydroxide reacts with dissolved CO,. Additionally, as sperm respire, acid is produced (Brokaw and Benedict, 1968). This acid release can be sufficiently rapid, particularly at higher temperatures, to
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
61
make measurement of the activation-associatedpH, changes difficult. The resultant recorder trace can appear as a small rapid change on top of much larger continuing acid efflux. Resolution of the rapid activation-dependentpH, changes is improved by reducing the temperature or by inhibiting mitochondria1 0, consumption with oligomycin (Hansbrough and Garbers, 1981) or cyanide (Schackmann et a l . , 1978; Lee, 1984a). The mixing chamber and electrode must be tested for their ability to respond to the appearance of acid in the extracellular medium. This is most easily performed by addition of a small aliquot of a strong acid such as HCI to a solution with and without sperm. The response of the electrode to the HCI addition should be considerably faster than that obtained upon activation of the sperm. The system we use gives responses about four-fold faster for HCI than to sperm acid release. Faster mixing is necessary for kinetic measurements.
B . Measurement of pH, Methods available for measurement of pH, include uptake of radiolabeled weak acids and bases, 3'P-nuclear magnetic resonance, and incorporation of molecular probes such as carboxyfluorescein which undergo spectral or fluorescent shifts with changes in pHi. Two excellent reviews on pH, measurements and methods have recently been published (Nuccitelli and Deamer, 1982; Busa and Nuccitelli, 1984). Since each probe for pHi may be subject to systematic error (examples are given below), the best experimental approach is to apply several methods to evaluate pHi. Agreement between unrelated methods is a strong indication that values determined are reliable.
1.
UFTAKE
OF
LABELED WEAKBASESAND ACIDS
a. Uptake of [I4C]Methylamine and [14C]Diethylamine. For reasons discussed below, uptake of weak bases has served as the primary method for measurement of pH, in sea urchin sperm. Uptake occurs because the uncharged, unprotonated amine rapidly equilibrates across the plasma membrane and is trapped when protonated within the cell (Schuldiner et al., 1972). Weak bases are concentrated by this mechanism into acidic environments. At high concentrations (10 mM is sufficient for sea urchin sperm at pH 8) uptake can increase pHi; at low concentrations it allows for measurement of pH, as intracellular buffering compensates for the small amount of amine accumulated. If a few specific conditions are satisfied,
pHi = pH, - log(Ar)
(1)
62
ROBERT SCHACKMANN
where Ar is the accumulation ratio, the apparent intracellular concentration of the weak base (or other probe molecule) divided by the extracellular concentration. For the simplest of conditions, measurement of pHi consists only of monitoring pH, and measuring the amount of weak base accumulated. Evaluation of uptake is performed by incubation of the sperm, typically a -100-fold dilution of dry sperm, with sufficient radioactive amine (-0.3 pCi/ml) until a steady state level of uptake is reached. Following rapid separation of the sperm from the reaction medium, radioactivity is counted and the Ar is determined (Schackmann et al., 1981). Several separation techniques have been employed. Centrifugation in a microfuge either directly or by layering an aliquot of the sperm suspension onto silicone oil gives values for pHi of -7.4 (Lee et al., 1983; Christen et al., 1982), in agreement with those of other techniques such as 31P-nuclearmagnetic resonance (31P-NMR)(Christen et al., 1983b). Assays are facilitated by spinning through silicone oil (Schackmann et al., 1981) as the pellets are then stable throughout the remainder of the experiment and do not require immediate attention. For sperm in artificial seawater (pH 8), pHi < pH, (Ar 2 -3), the amount of radioactive weak base trapped in the extracellular space is therefore
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
63
for example, of 9-aminoacridine (Christen et al., 1982). Also, it has been calculated that increasing the ApH (i.e., increasing Ar) increases the time required for equilibrium to be attained (Christen et al., 1982). b. Uptake of ('4C]DM0. Uptake of the weak acid dimethyloxazolidine-2,4-dione (DMO) has also been used as a measure of pH, in sperm (Schackmann et a/., 1981). Unlike weak bases, weak acids accumulate into regions of higher pH as the unprotonated acid is charged (Waddell and Butler, 1959). An Ar of -0.5 for [14C]DM0 in sperm in artificial seawater (pH 8) gives an apparent pH, of 7.7. The difference between this value and that for weak bases (pHi -7.4) can be explained by uptake of the weak acid into the mitochondrion. Mitochondria are usually basic with respect to the cytoplasm (Hoek et al., 1980). In sea urchin sperm, the single mitochondrion may occupy a volume of up to 30% of the total intracellular space (Harvey and Anderson, 1943; Schackmann et al., 1984), hence it is a substantial fraction of the intracellular volume. If the intramitochondrial space pH is 8.0, then uptake of both diethylamine (Ar - 5 ) and DMO (Ar -0.5) is consistent with a value for the cytoplasm pH, of -7.3. A complete, detailed analysis of the problem of subcellular compartments of different pH has been provided by Hoek and colleagues (1980). As calculated by Christen and colleagues (1982), at pH, 8 only -7% of the weak base partitions into the mitochondrion whereas as much as 80% of the weak acid may be located there. In sea urchin sperm in seawater at pH 8 weak bases therefore provide a more direct indicator of the cytoplasm pH,. c. Uptake of9-Arninoacridine. An alternative to measurement of pH, with radioactive weak bases involves measurement of fluorescence quenching of 9aminoacridine. 9-Aminoacridine is a weak base and accumulates by diffusion of the unprotonated amine (Schuldiner et al., 1972; Christen et al., 1982). It has the property that its fluorescence is highly quenched upon uptake by sperm. Measurement of fluorescence quenching is equivalent to following disappearance of the 9-aminoacridine from the seawater as it is accumulated. Up to 80% of the dye may be taken up, yielding Ar values of 2200 or larger (Schackmann et al., 198I ; Christen er al., 1982). If a pH, value were determined directly from Eq. ( l ) , a pH, of -4.7 would be calculated, in complete disagreement with values for other weak bases. However, a careful study revealed that most acridine uptake occurs as a result of the intracellular binding of the dye following accumulation as weak base (Christen et al., 1982). That is, even if ApH is completely collapsed with detergents or ionophores, substantial uptake remains. The binding properties of the sperm for 9-aminoacridine were studied and it was found that at an initial external concentration of 1-2 pM, intracellular binding was far from saturation. As explained in detail in the supplement to the paper by Christen et al. (1982), this allows for correction of the binding in a simple manner to give the following equation:
64
ROBERT SCHACKMANN
where Q is fraction of fluorescence quenched (fraction of 9-aminoacridine taken up) and Ql is the fraction quenched in the presence of either detergent or ionophores to collapse ApH across the plasma membrane. The unexpected advantage of this method of pHi measurement is that intracellular water space need not be measured. With this correction pH, values determined by uptake of 9aminoacridine agree well with those determined by uptake of [ 14C]diethylamine or with 31P-NMR(Christen er al., 1982, 1983b). 2. pH, MEASUREMENTS BY 31P-NMRAND
WITH
CARBOXYFLUORESCEIN
Two measurements of sea urchin sperm pHi by 31P-NMRhave been reported (Christen et al., 1983b; Johnson er al., 1983). Measurement of the chemical shift of the 31Piis directly related to pHi (for review, see Nuccitelli and Deamer, 1982). The chemical shift of Pi varies with the degree of protonation of Pi and is a good indicator of pHi in the vicinity of the pK of Pi. Measurement of pHi therefore necessitates a pH calibration of the Pi chemical shift within the sperm. Christen and colleagues (1983b) approached this problem by collapsing the ApH with the ionophores nigericin and monensin so that pHi = pH,. Varying pHe and recording the Pi chemical shift allows for a calibration within the intracellular environment. Values determined by 31P-NMRin sperm are in excellent agreement with those determined by uptake of diethylamine (Christen et al., 1983b). For example, at pHe8 for sperm in artificial seawater, the pH, was 7.4 by 31PNMR, 7.3 by [ 14C]diethylamineaccumulation, and 7.4 by 9-aminoacridine uptake (Christen et al., 1982). And for quiescent sperm in Na+-free seawater, pH, was 6.8 by 31P-NMR and 6.9 by [14C]diethylamine uptake. A limitation of NMR is the requirement for high cell concentration. A 10% sperm suspension was necessary to yield a satisfactory spectrum in a reasonable amount of time (minutes). This precludes time-dependent resolution of pH, changes which are complete in seconds. However, 31P-NMRdoes provide the only reasonable method of analyzing the pHi in dry sperm before dilution. A pH, value of -7 was obtained, which is sufficiently low to explain sperm immotility in semen (Johnson er af., 1983; Christen er al., 1983b). Carboxyfluorescein and other optical pH probes have yet to be used as pHi probes for sea urchin sperm. A detailed report of carboxyfluorescein use for measurement of pHi in bovine sperm has been published by Babcock (1983) and the interested reader is referred to this article. It is mentioned here because intracellular optical probes provide the possibility of real-time resolution of the pHi changes important to sperm activation. For sea urchin sperm, measurement of pHi by following weak base uptake is
4.
65
ION MEASUREMENTS IN SEA URCHIN SPERM
the most direct method and can be performed without elaborate equipment. Uptake reflects the pH, of the apparent “cytoplasm” since it is in agreement with values determined by 31P-NMR and the bases will not accumulate into the mitochondria. It remains to be seen whether weak base uptake will be useful for evaluation of pH, in sperm of other species. The large acrosomal region of mammalian sperm has led Meizel and Deamer (1978) to propose that 9-aminoacridine uptake measures the intraacrosomal pH in hamster sperm.
IV. Measurement of Cation Uptake In the previous section pH, was discussed as it is an important regulatory component of sperm motility and the acrosome reaction. pH, changes in sea urchin sperm correlate with or are linked with other cation movements as well. In particular, a Na / H exchange is a component of the sperm plasma membrane that can either raise or lower the pH, (Lee, 1984a,b). Furthermore, induction of the acrosome reaction results in multiple changes in the sperm cation composition (Schackmann et al., 1981; Cantino ef al., 1983). Na+ and Ca2+ enter the sperm and K (as well as H +) exit. Of these Ca2 entry is a crucial requirement for the acrosome reaction regardless of the method used to initiate it. +
+
+
A.
+
Measurement of Ca*+ 1. 4sCa2+ UFTAKE
The most frequently used method for measurement of Ca2+ entry into sperm consists of following 45Ca2+ accumulation. The basic methodology is the same as that for monitoring uptake of weak bases (Section III,B,l ,a). Isotopic 4sCa2+ is added to the seawater and incubated with the sperm under the experimental conditions to be tested. Termination of uptake is effected by rapid separation of the sperm from the medium by either centrifugation or filtration. Most experiments have been performed by filtration (Schackmann et al., 1978; Kopf et al., 1984; Kopf and Garbers, 1980). I have found that centrifugation of sperm through silicone fluid works equally well, although background 45CaZ uptake is higher (-4 nmol/108 sperm) than for filtration (-1 nmol/108 sperm). Whether this represents rapid Ca2+ uptake, CaZ+ binding to the surface, or both, is not known. When the acrosome reaction is triggered, increased 45Ca2+ uptake is initiated and may reach -20 nmol/108 sperm (Schackmann et al., 1978). Such an increase is easily resolved by either filtration or centrifugation. Stimulation of 4sCa2 entry indicates that enhanced Ca2 influx occurs but does +
+
+
66
ROBERT SCHACKMANN
not provide direct evidence that net Ca2+ uptake results. In order to show that a net increase occurred, atomic absorption spectroscopy and X-ray microanalysis were performed. I will not deal with the methodology directly but refer the reader to work by Cantino and colleagues (1983). The results obtained by either method show that net Ca2+ uptake does correspond to 45Ca2+ uptake. X-Ray microanalysis also allowed for localization of the Ca2 uptake to the mitochondria as predicted from inhibitor studies (Schackmann and Shapiro, 1981). The slow, long-term accumulation of Ca2 for -30 minutes after the acrosome reaction is complete represents a complex function of Ca2 entry through the plasma membrane and accumulation by the mitochondrion. An additional factor limiting the usefulness of 45Ca2+ uptake is the level of resolution attained. With a background level of 1-4 nmol/108 sperm, it is extremely difficult to resolve increases of even 0.1 nmol/ lo8 sperm as were found when the acrosome reaction was initiated in the presence of drugs to inhibit mitochondria1 Ca2+ uptake (Schackmann and Shapiro, 1981). Yet a change of this magnitude could still represent an extremely large change in [Ca2+Ii.A need for measurement of changes in free [Ca2+Iiis clearly indicated. +
+
+
2.
MEASUREMENT OF [Ca2+Ii
Tsien et al. (1982) and colleagues (Grynkiewicz et al., 1985) have developed several Ca2+ indicator dyes which show altered fluorescence upon binding of Ca2 . These dyes can be loaded into cells as uncharged molecules if the carboxyl groups involved in binding Ca2+ are first esterified. The ester groups are hydrolyzed within cells to generate the native Ca2+ indicators. These dyes, “Quin2” (Tsien er al., 19821, “fura-2,” and “indo-1” (Grynkiewicz et al., 1985), have dissociation constants of 100 nM and are sufficiently insensitive to [Mg2+] and [H+]to allow for measurement of [Ca2+] within living cells. As highly charged anions, they do not readily leak from cells. These dyes have only recently begun to be applied to sperm physiology. In an experiment described in Table I, nigericin, a monovalent cation inonophore that triggers the acrosome reaction and stimulates 45Ca2+ uptake (Schackmann et al., 1978), was found to increase Quin2 fluorescence. The data indicate that [Ca2 ] increases approximately 8-fold within a minute following addition of the nigericin. Further application of these dyes to sperm physiology will allow for a more complete understanding of [Ca2+ I i changes important to sperm activation. +
-
+
B. Measurement of Na+ and K + The simplest method for measurement of Na+ or K + uptake is to follow 22Na+ or 42K fluxes. 42K has a 12-hour half-life and 86Rb has been used as a K + substitute (Schackmann et al., 1978). Recent attention has focused on +
+
+
4.
67
ION MEASUREMENTS IN SEA URCHIN SPERM
TABLE I NlGERlClN INCREASES
Conditions ASW. 5 mM Ca2+ + nigericin (60 phf) ASW, 0 mM Ca2+ + nigericin (60 phf) ASW, 5 mM Ca2+ + ionomycin (30 phf) + ionomycin + 3 mM Mn2+
[Ca2+]t'
Fluorescence (arbitrary units)
(Ca2 Ii (units of K d )
43 91 41 40
0.30 2.3 0.27 0.25
I22 0
-
+
Sperm (10% dry sperm by volume) of the sea urchin, S. purpuratus, were incubated 5 hours in calcium-free seawater, pH 6.8, with I mM EGTA and 50 phf Quin2-acetoxymethyl ester (Tsien et a / ., 1982). After washing the sperm and resuspending the pellet as 20% dry sperm, 100 pl was added to I .9 ml ASW at the indicated [Ca2+]. Ionophores were followed by I-minute incubations prior to fluorescence measurements. [Ca2+] was calculated as ( F F,,,/F,,,, - F)Kd (Tsien et a / ., 1982). where F = fluorescence and F,,, = the fluorescence with 5 mM Ca2+ and ionomycin. FIni,= 0.16 AF where A F is between F,,, and the F with ionomycin + Mn2+ to quench Quin2 fluorescence (Hesketh el a / . . 1983). Kd is the dissociation constant for the Quin2Ca2f complex within the sperm.
22Na+ movements because Na+ / H + exchange has been linked to pH, changes associated with motility activation (Lee, 1984a; Hansbrough and Garbers, 1981). Uptake of 22Na+ is complete within seconds when motility and respiration are stimulated by Na+ addition to sperm in Na+-free seawater (Lee, 1984a; Schackmann, unpublished data). 22Na+ entry is rapid when the acrosome reaction (Schackmann and Shapiro, 1981) is triggered and is sufficiently rapid in intact sea urchin sperm tails (Lee, 1984a,b) to preclude a kinetic analysis. These studies of 22Na uptake were performed by filtration on glass fiber filters (Whatman GF/C). As noted in Section IV,A, 1, for 45Ca2 uptake, both centrifugation and filtration gave comparable results. However, centrifugation of the sperm through silicone oil gives values of 22Na uptake approximately two-fold higher than by filtration (Cantino et af., 1983) and these values are in much closer agreement to those determined by X-ray microanalysis. Presumably, when sperm are filtered and washed, some 22Na+ exits rapidly. The time taken for 22Na+ to equilibrate across the sperm membranes is about the same magnitude as the time necessary for washing the filters and could lead to underestimates for Na uptake. Such apparent differences demand careful attention when quantifying Na+ uptake. The recently described N a + / H + exchange in isolated tails from sea urchin sperm is thought to be electrically neutral (Lee, 1984b) on the +
+
+
+
68
ROBERT SCHACKMANN
basis of an apparent 1:1 stoichiometry. Since different techniques yield different values for Na uptake, arguments for electroneutrality based on filtration measurements alone are unwarranted without careful substantiation. Even larger differences between data acquired by filtration and centrifugation have been registered for K uptake (Cantino et al., 1983). Again, centrifugation yields values in closer agreement to those determined by atomic absorption or Xray microanalysis. An additional difficulty exists with 22Na+ measurements. Evaluation of ion uptake is difficult if the ion is not concentrated within the sperm; that is if Ar 5 1. This means that measurement of K is quite simple since the Ar, + = -20 but for sperm in artificial seawater ATNa+= -0.3. The amount of Na trapped in the extracellular space is at least 50% of the total amount pelleted with the sperm. At normal levels of seawater Na+ both methods, filtration and centrifugation, are experimentally difficult to use as accurate quantitative measurements for Na+ uptake. +
+
+
+
V . Measurement of Membrane Potentials in Sperm The small size of sea urchin sperm has precluded attempts at measurement of membrane potentials with conventional microelectrodes. Measurements on intact sperm have relied on uptake of the diffusable lipophilic ions [ 14C]thiocyanateand [3H]tetraphenylphosphonium+(TPP+) (Schackmann et al., 1981, 1984). This method was designed to measure membrane potentials in bacteria and in membrane vesicles where electrode technology was not applicable (Lichtshtein et al., 1979; Rottenberg, 1979). If a negative membrane potential exists, the TPP+ cation will accumulate and the thiocyanate anion will be excluded (Ar < 1). We verified this for sperm of the sea urchin Strongylocentrotus purpuratus. Additionally, [3H]TPP+ uptake was decreased by increasing seawater [K+ 1, an event that is consistent with the depolarization of the plasma membrane potential by elevated extracellular [K 1. Conversely, [ I4C]thiocyanate uptake increased with increasing seawater [K+]. Qualitatively these data imply that the sea urchin sperm plasma membrane potential is similar to that of many other cells. Quantitative evaluation of the potentials with these membrane potential probes is more difficult, particularly for TPP+ , which largely accumulates into the mitochondrion (Schackmann et a f . , 1984) by virtue of the large negative mitochondrial potential. We were able to explore the contribution of mitochondria1 potential in intact sperm using [14C]thiocyanate ([ I4C]SCN) to measure the plasma membrane potential and using [3H]TPP to measure a composite potential that is dependent upon the two negative potentials acting in series. Additionally, by making the plasma membrane permeant with digitonin, we were able +
+
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
69
to generate with an exogenous substrate a mitochondria1potential consistent with our in vivo measurements (- 155 mV). If corrections are made for the mitochondrial accumulation of TPP+ , both TPP+ and SCN yield values of the plasma membrane potential of -30 mV for sperm in artificial seawater. [14C]SCN uptake in principle provides a more direct measurement of the plasma membrane potential; but since Ar < 1, the error in these measurements is large. Measurements of [3H]TPP+ and ['4C]SCN uptake have been made principally by centrifugation through silicone oil (Schackmann et al., 1984). TPP+ was found to have limited solubility in the silicone oil. Diffusion from the sperm pellet was not substantial within 24 hours, but diffusion from the supernate into the silicone oil layer was found to erroneously enhance the value for Ar (supernatant counts decreased). Samples should, therefore, be processed immediately following an experiment. Although we have used the lipophilic ions to provide the first values for membrane potentials in sea urchin sperm, the methods have serious limitations. The kinetics of TPP+ uptake into sperm are extremely slow, thus only static, long-term charges in potential can be observed. It has proved possible to measure a depolarization of -30 mV when the acrosome reaction is induced. But if transient changes occurred prior to the net depolarization, we would be unable to detect them. And like uptake of weak bases, the time-course for changes in uptake does not reflect the time-course for changes in A+, but reflects a complex function of the potentials and sperm permeability to the probe molecules. Recent advances in patch-clamping small sections of cell membranes (Neher et al., 1978) should ultimately allow more exact, rapid measurements of sperm membrane potentials. In this regard, LiCvano and colleagues (1985) have only recently reported making lipid bilayers of sea urchin sperm membranes on a patch electrode. They found K + channels which could be blocked by tetraethylammonium. This potentially represents an exciting new method of analyzing ion movements through sperm membranes.
-
VI. Conclusion I have briefly attempted to provide the reader interested in measuring ionic changes in sperm with a perspective on how the measurements have been and should be made and the potential limitations inherent with existing techniques. Simple ionic manipulation, regulation of pH,, and [Ca2+] are of fundamental importance to sperm physiology. Investigations of ionic changes associated with activation of sea urchin sperm have allowed us to begin to define how these important events are initiated and lead to activation of development. Whether these changes are unique to echinoid sperm or are general characteristics of
70
ROBERT SCHACKMANN
sperm remains to be shown. Similar ionic changes, increases in pHi, and [Ca2+Ii are known to attend to a host of cell activation events throughout the animal kingdom. The apparent similarity between echinoid sperm activation and activation of other eukaryotic cells raises additional interest in the study of echinoid sperm and the ionic changes important to their activation. ACKNOWLEDGMENTS I wish to thank Merry Peters for typing this manuscript. This work was supported by Grant HD 18670 from the National Institutes of Health.
REFERENCES Babcock, D. F. (1983). J . Biol. Chem. 258, 6380-6389. Bibring, T., Baxandall, J., and Harter, C. C. (1984). Dev. Biol. 101, 425-435. Boyer, P. D., Chance, B., Emster, L., Mitchell, P., Racker, E., and Slater, E. C. (1977). Annu. Rev. Biochem. 46, 955-1026. Brokaw, C. J., and Benedict, B. (1968). J. Gen. Physiol. 52, 283-299. Busa, W. B., and Nuccitelli, R. (1984). Am. J . Physiol. 246, R409-438. Cantino, M. E., Schackmann, R. W., and Johnson, D. E. (1983). J . Exp. Zool. 226, 255-268. Christen, R., Schackmann, R. W., and Shapiro, B. M. (1982). J. Biol. Chem. 257, 14881-14890. Christen, R., Schackmann, R. W., and Shapiro, B. M. (1983a). J . Biol. Chem. 258, 5392-5399. Christen, R., Schackmann, R. W., Dahlquist, F. W., and Shapiro, B. M. (1983b).Exp. Cell Res. 149, 289-294. Dan, J. C. (1952). Biol. Bull. (Woods Hole) 103, 54-56. Dan, J. C. (1954). Biol. Bull. (Woods Hole) 107, 335-349. Grynkiewicz, G., Poenie, M., and Tsien, R. Y.(1985). J. Biol. Chem. 260, 3440-3450. Hansbrough. J. R., and Garbers, D. L. (1981). J . Biol. Chem. 256, 2235-2241. Harvey, E. B., and Anderson, T. F. (1943). Biol. Bull. (Woods Hole) 85, 151-156. Hesketh, T. R., Smith, G. A., Moore, J. P.,,Taylor, M. V., and Metcalfe, J. C. (1983). J. Biol. Chem. 258,4876-4882. Hesketh, T. R., Moore, J. P., Moms, J. D. H., Taylor, M. V., Rogers, J., Smith, G. A., and Metcalfe, J. C. (1985).Nature (London) 313, 481-484. Hoek, I. B., Nicholls, D. G., and Williamson, J. R. (1980). J. Biol. Chem. 255, 1458-1464. Johnson, C. H., Clapper, D. L., Winkler, M. M., Lee, H. C., and Epel, D. (1983).Dev. Biol. 98, 493-501. Johnson, J. D., Epel. D., and Paul, M. (1976). Narure (London) 262, 661-664. Kopf, G. S., and Garbers, D. L. (1980).Biol. Reprod. 22, I I 18-1 126. Kopf, G., Lewis, C. A,, and Vacquier, V. D. (1984). J . Biol. Chem. 259, 5514-5520. L’Allemain, G., Pans, S., and PouyssCgur, J. (1984). J . Biol. Chem. 259, 5809-5815. Lee, H. C. (1984a).J . Biol. Chem. 259, 4957-4963. Lee. H. C. (1984b). J . Biol. Chem. 259, 15313-15319. Lee, H. C., Johnson, C., and Epel, D. (1983). Dev.Biol. 95, 31-45. Lichtshtein, D., Kaback, H. R., and Blume, A. J. (1979).Proc. Narl. Acad. Sci. U . S . A . 76, 650654. LiCvano, A., Shchez, J., and Darszon, A. (1985). Biophys. J . 47, (2, Pt. 2), 145a. Madeira, V. M. C. (1975). Biochem. Biophys. Res. Commun. 64, 870-876. Meizel, S., and Deamer, D. W. (1978). J. Hisrochem. Cyrochem. 26,98-105.
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Moolenaar, W. H., Tertoolen, L. G . 1.. and de Laat, S. W. ( I 984). J. Biol. Chem. 259,7563-7569. Moore, R. A., Fidelman, M. L., Hansen, J. C., and Otis, J. N. (1982). In “Intracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions” (R. Nuccitelli and D. W. Deamers, eds.), pp. 386-416. Liss, New York. Neher, E., Sakmann. B., and Steinbach. J. H. (1978). Pfliigers Arch. Ges. Physiol. 375, 219-228. Nishioka, D., and Cross, N. (1978).In “Cell Reproduction” (E. R. Dirksen, D. Prescott, and D. F. Fox, eds.), pp. 403-413. Academic Press, New York. Nuccitelli. R., and Deamer, D. W. (1982). “lntracellular pH: Its Measurement, Regulation, and Utilization in Cellular Functions” (R.Nuccitelli and D. W.Deamers, eds.). Liss, New York. Repaske, D. R., and Garbers. D. L. (1983). J . Biol. Chem. 258, 6025-6029. Ridgway, E. B . , Gilkey, 1. C., and Jaffe, L. F. (1977). Proc. Nail. Acad. Sci. U.S.A. 74,623-627. Rothenberg, P., Reuss. L., and Glaser, L. (1982). Proc. Nail. Acad. Sci. U.S.A. 79, 7783-7787. Rothenberg, P., Glaser, L., Schlesinger, P., and Cassel, D. (1983). J . Biol. Chem. 258, 1264412653. Rottenberg, H. (1979).In “Methods in Enzymology” (S. Fleisher and L. Packer, eds.). Vol. 55, pp. 547-569. Academic Press, New York. Schackmann, R. W., and Shapiro, B. M. (1981). Dev. Biol. 81, 145-154. Schackmann, R. W., Eddy, E. M., and Shapiro, B. M. (1978). Dev. Biol. 65, 483-495. Schackmann, R. W., Christen, R., and Shapiro, B. M. (1981). Proc. Nail. Acad. Sci. U.S.A. 78, 6066-6070. Schackmann, R. W., Christen, R., and Shapiro, B. M. (1984).J . Biol. Chem. 259, 13914-13922. Schroeder, T. E., and Christen, R. (1982). Exp. Cell Res. 140, 363-371. Schuldiner, S., Rottenberg. H., and Avron. M. (1972). Eur. J . Biochem. 25, 64-70. Shen, S . S . , and Steinhardt, R. A. (1978). Nuturr (London) 272, 253-254. Steinhardt, R.. Zucker. R . , and Schatten, G. (1977). Dab. Biol. 58, 185-196. Thomas, A. P., Alexander, J., and Williamson, J. R. (1984). J. Biol. Chem. 259, 5574-5584. Tilney, L. G . , Kiehart. D. P., Sardet, C., and Tilney, M. (1978). J . Cell Biol. 77, 536-550. Tsien, R. Y., Pozzan, T., and Rink, T. J. (1982). J. Cell Biol. 94, 325-334. Waddell, W. J . , and Butler, T. C. (1959). J . Clin. Invest. 38, 720-729.
1. 11.
HI. IV . V. VI.
............
............
A. Sperm Preparation .......................... B. Measurement of lntracellular and Extracellular Water Space . . . . . . . . . . . . . . . pH Measurements Important to Sperm Activation ............................ A. Measurement of Acid Efflux ......................................... B. Measurement of pHi ................ Measurement of Cation Up A. Measurement of Ca2+ ....................... B. Measurement of Na+ and K + ................ . . . . . . . . . . . . . . . . . . . . . . . Measurement of Membrane .............. Conclusion . . . . . . . . . . . . . .............. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I.
57 58 58
59 60 60 61
66 68 69 70
Introduction
Recent literature, both biochemical and physiological, pays increasing attention to relationships among cellular ion movements, ion-translocating systems, and cellular function. Ion transport, once thought to be specific for tissues containing excitable cells or cells specialized for salt or acid secretion, appears now to be a component of cell responses to stimuli throughout growth and development. Na+ / H + exchanges, which increase pHil (Johnson et al., 1976; Shen and Steinhardt, 1978; Hesketh et al., 1985; L'Allemain et al., 1984; Moolenaar er al., 1984; Rothenberg er al., 1983), changes in [Ca2+Ii(Ridgway 'Abbreviations: pHi, intracellular pH; pH,, extracellular pH; [Ca* I,, intracellular calcium concentration; Ar, accumulation ratio; TPP, tetraphenylphosphonium; SCN, thiocyanate; DMO, 5.5dimethyloazolidine-Z,4-dione;P,, inorganic phosphate. +
57 METHODS IN CELL BIOLOGY. VOL. 27
Copyright 0 1986 by Academic R s s . Inc. All rights of reproduction in any form reserved.
58
ROBERT SCHACKMANN
et al., 1977; Steinhardt er al., 1977; Hesketh et al., 1985), or changes in the plasma membrane potential (Rothenberg et al., 1982) are among the earliest responses of quiescent eukaryotic cells to stimulation of cell growth. Cellular responses to a variety of hormonal agonists have been linked to both alterations of [Ca2+Ii (Thomas et af., 1984) and pHi (Moore et al., 1982), and ATP production by oxidative phosphorylation (Boyer et al., 1977) involves pH and potential gradients operating at the subcellular level. These are but a few examples of biological systems in which ions serve as regulatory components of cell biology. Historically, echinoid sperm were among the first cells used to provide evidence that ionic changes are important to cell activation. It was demonstrated 3C years ago by Jean Dan that the sea urchin sperm underwent an acrosomal reaction when exposed to factors released from the egg (egg jelly). This reaction was defined morphologically to consist of exocytosis of the acrosomal granule and polymerization of actin into the acrosomal rod. Dan (1954) showed that removal of Ca2 from the seawater prevented the acrosomal reaction. Likewise increasing the pH, to 9.2 spontaneously initiated the acrosomal reaction in the absence of any of the egg factors (Dan, 1952). Thus, ionic alterations were implicated in the activation of the sperm for fertilization. We now know that egg factors that initiate the acrosomal reaction cause both Ca2+ influx and increased pHi (Schackmann et al., 1978, 1981). The Ca2+ influx is thought to lead to exocytosis of the acrosomal granule while increased pH, can cause actin polymerization (Tilney et al., 1978; Schroeder and Christen, 1982). Additionally, pH, is a primary regulatory component of sea urchin sperm motility and respiration (Christen et al., 1982, 1983a,b; Lee et al., 1983). At low pH, (< -7.0) sperm are immotile. As pH, is increased to the normal pHi of -7.4, sperm respiration and motility are initiated. These findings underlie the importance for studying ion movements as regulatory components of sperm activation. This article discusses methods used to measure ion changes important to activation of sea urchin sperm motility and the acrosome reaction. I describe briefly the methods used by several laboratories to measure pH,, ion fluxes, and membrane potentials in sea urchin sperm. It is not my intention to explain in complete detail how to make the ion measurements, but to provide a supplement to published methods which hopefully contains additional insight on their application. +
11. A.
Sperm Preparation and Determination of Intracellular Water Space
Sperm Preparation
Sperm are typically collected from sea urchins by injecting a few milliliters of 0.5 M KCl into the coelomic cavity. After initiation of spawning, the animals are
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
59
inverted and allowed to shed into a Petri dish without seawater (“dry sperm”). Experiments are usually performed at 0.1 to 10% suspensions of “dry sperm” (vo1ume:volume). The supernatant of centrifuged dry sperm has been analyzed by atomic absorption spectroscopy (Johnson et al., 1983) and except for [ K + ] (20-30 mM), the composition approximates that of seawater. Thus “a+] is > 400 mM and [Ca2+] is -10 mM. The high “a+] necessitates that sperm be washed, if they are to be removed from Na+ . A 100-fold dilution contains -4 mM Na . This is sufficient Na to activate sperm motility and respiration (Christen et al., 1982). Washing of sperm is easily accomplished by gentle centrifugation at IOOO-1500 g for -10 minutes. +
+
B . Measurements of Intracellular and Extracellular Water Space It is convenient to measure uptake and cell content of ions, expressed per unit of cell protein or cell number. However, interpretation of the physiological significance of the ion content often requires an approximation of the intracellular concentration, which requires evaluation of the intracellular water space. The accepted method for evaluating intracellular water consists of a difference measurement between total [3H]H20 in a pellet of cells and the pellet volume occupied by molecules impermeant to the plasma membrane (Rottenberg, 1979). Water rapidly penetrates biological membranes and the volume of [3H]H,0 in a pellet represents the total of intra- and extracellular water space. Typically, sugar molecules fill the role as impermeant, extracellular markers. [ ‘‘C]Inulin, sucrose, and sorbitol have all been used with sea urchin sperm (Schackmann et al., 1981; Lee et al., 1983). Values for the extracellular space are -0.2 pl/IO* sperm. The intracellular water space is 0.7-0.8 pl/ lo* sperm (Schackmann et al., 198 I ; Lee et ul., 1983) and is quite close to the morphologically determined value of 0.72 p1/ lo* sperm for the sea urchin A. punctulatu (Harvey and Anderson, 1943). Sucrose is easier to use than carboxy-inulin as the latter tends to accumulate precipitative radioactive material which can lead to erroneously large volumes for extracellular space (in extreme cases exceeding the total [”H]H2O space). However, sucrose is finitely permeant and incubations as an extracellular marker should be kept to only a few minutes. Since the intracellular H 2 0 space measurement is determined by a difference between experimental values, each with its own inherent experimental error, the total error in the intracellular water content can be larger than that for measurement of uptake of the ions or pHi probes themselves. Small differences or changes in intracellular volume are also not easily detected. It is clearly important to evaluate the cell and pellet water spaces under each set of experimental conditions used. The accuracy of the calculations of ion concentrations, pH,, or membrane potential are no better than the water space measurements themselves.
60
ROBERT SCHACKMANN
111. pH Measurements Important to Sperm Activation Initiation of motility and induction of the acrosome reaction are associated with increases in pH, regardless of the stimulus applied. To be motile the sperm pH, must be 2 -7.2 (Christen et af., 1982, 1983a,b; Lee et af., 1983). For the acrosome reaction to occur the pHi must be raised to -7.6 (Schackmann et af., 1981). However, the experiments first indicating the importance of pHi increases in sperm activation were not based on direct measurements of pHi, but rather were observations that acid efflux occurred upon activation of either motility or the acrosome reaction. Activation of sperm respiration and motility by dilution (Johnson et af., 1983), by addition of the egg peptide “speract” (Hansbrough and Garbers, 1981; Repaske and Garbers, 1983), or by addition of Na+ to sperm in Na+-depleted seawater (Nishioka and Cross, 1978; Lee, 1984a), all result in acid efflux and elevation of pHi. Induction of the acrosome reaction by either egg jelly or ionophores was likewise found to be accompanied by acid efflux (Schackmann et af., 1978; Tilney et af., 1978).
A.
Measurement of Acid Efflux
Measurement of the acid efflux upon activation of motility or the acrosome reaction provides rapid quantitative information necessary to understand how H + efflux is coupled to other ion movements such as Na+ entry (see Section IV,B). To measure the efflux a pH electrode is placed in a sperm suspension, agents are added to stimulate the sperm, and changes in the external pH are followed with a recorder. For example, when Na+ is added to a 1% sperm suspension in Na+-free seawater, H + efflux of -100 nmol occurs. The resulting pH, decrease is easily monitored with most of the better modem pH meters and electrodes. Our laboratory uses a pHM64 Radiometer pH meter with a GK2310-C combination electrode. The output to the recorder must be offset (Madeira, 1975). With seawater buffered with HC0,- (Lee, 1984a) or low concentrations of Tris or HEPES (- 1 mM), variation of the sperm concentration allows manipulation of the total external pH change. I try to keep the change to -0.05 pH units to approximate measurements at a constant pH,. The response time of the electrode must be reasonably fast as the rapid pH changes due to activation of the acrosome reaction and motility initiation are complete within 15 seconds. Calculation of acid equivalents released is usually performed by titration with freshly prepared NaOH or KOH. Stock solutions should be standardized against an acid of known concentration since hydroxide reacts with dissolved CO,. Additionally, as sperm respire, acid is produced (Brokaw and Benedict, 1968). This acid release can be sufficiently rapid, particularly at higher temperatures, to
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
61
make measurement of the activation-associatedpH, changes difficult. The resultant recorder trace can appear as a small rapid change on top of much larger continuing acid efflux. Resolution of the rapid activation-dependentpH, changes is improved by reducing the temperature or by inhibiting mitochondria1 0, consumption with oligomycin (Hansbrough and Garbers, 1981) or cyanide (Schackmann et a l . , 1978; Lee, 1984a). The mixing chamber and electrode must be tested for their ability to respond to the appearance of acid in the extracellular medium. This is most easily performed by addition of a small aliquot of a strong acid such as HCI to a solution with and without sperm. The response of the electrode to the HCI addition should be considerably faster than that obtained upon activation of the sperm. The system we use gives responses about four-fold faster for HCI than to sperm acid release. Faster mixing is necessary for kinetic measurements.
B . Measurement of pH, Methods available for measurement of pH, include uptake of radiolabeled weak acids and bases, 3'P-nuclear magnetic resonance, and incorporation of molecular probes such as carboxyfluorescein which undergo spectral or fluorescent shifts with changes in pHi. Two excellent reviews on pH, measurements and methods have recently been published (Nuccitelli and Deamer, 1982; Busa and Nuccitelli, 1984). Since each probe for pHi may be subject to systematic error (examples are given below), the best experimental approach is to apply several methods to evaluate pHi. Agreement between unrelated methods is a strong indication that values determined are reliable.
1.
UFTAKE
OF
LABELED WEAKBASESAND ACIDS
a. Uptake of [I4C]Methylamine and [14C]Diethylamine. For reasons discussed below, uptake of weak bases has served as the primary method for measurement of pH, in sea urchin sperm. Uptake occurs because the uncharged, unprotonated amine rapidly equilibrates across the plasma membrane and is trapped when protonated within the cell (Schuldiner et al., 1972). Weak bases are concentrated by this mechanism into acidic environments. At high concentrations (10 mM is sufficient for sea urchin sperm at pH 8) uptake can increase pHi; at low concentrations it allows for measurement of pH, as intracellular buffering compensates for the small amount of amine accumulated. If a few specific conditions are satisfied,
pHi = pH, - log(Ar)
(1)
62
ROBERT SCHACKMANN
where Ar is the accumulation ratio, the apparent intracellular concentration of the weak base (or other probe molecule) divided by the extracellular concentration. For the simplest of conditions, measurement of pHi consists only of monitoring pH, and measuring the amount of weak base accumulated. Evaluation of uptake is performed by incubation of the sperm, typically a -100-fold dilution of dry sperm, with sufficient radioactive amine (-0.3 pCi/ml) until a steady state level of uptake is reached. Following rapid separation of the sperm from the reaction medium, radioactivity is counted and the Ar is determined (Schackmann et al., 1981). Several separation techniques have been employed. Centrifugation in a microfuge either directly or by layering an aliquot of the sperm suspension onto silicone oil gives values for pHi of -7.4 (Lee et al., 1983; Christen et al., 1982), in agreement with those of other techniques such as 31P-nuclearmagnetic resonance (31P-NMR)(Christen et al., 1983b). Assays are facilitated by spinning through silicone oil (Schackmann et al., 1981) as the pellets are then stable throughout the remainder of the experiment and do not require immediate attention. For sperm in artificial seawater (pH 8), pHi < pH, (Ar 2 -3), the amount of radioactive weak base trapped in the extracellular space is therefore
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
63
for example, of 9-aminoacridine (Christen et al., 1982). Also, it has been calculated that increasing the ApH (i.e., increasing Ar) increases the time required for equilibrium to be attained (Christen et al., 1982). b. Uptake of ('4C]DM0. Uptake of the weak acid dimethyloxazolidine-2,4-dione (DMO) has also been used as a measure of pH, in sperm (Schackmann et a/., 1981). Unlike weak bases, weak acids accumulate into regions of higher pH as the unprotonated acid is charged (Waddell and Butler, 1959). An Ar of -0.5 for [14C]DM0 in sperm in artificial seawater (pH 8) gives an apparent pH, of 7.7. The difference between this value and that for weak bases (pHi -7.4) can be explained by uptake of the weak acid into the mitochondrion. Mitochondria are usually basic with respect to the cytoplasm (Hoek et al., 1980). In sea urchin sperm, the single mitochondrion may occupy a volume of up to 30% of the total intracellular space (Harvey and Anderson, 1943; Schackmann et al., 1984), hence it is a substantial fraction of the intracellular volume. If the intramitochondrial space pH is 8.0, then uptake of both diethylamine (Ar - 5 ) and DMO (Ar -0.5) is consistent with a value for the cytoplasm pH, of -7.3. A complete, detailed analysis of the problem of subcellular compartments of different pH has been provided by Hoek and colleagues (1980). As calculated by Christen and colleagues (1982), at pH, 8 only -7% of the weak base partitions into the mitochondrion whereas as much as 80% of the weak acid may be located there. In sea urchin sperm in seawater at pH 8 weak bases therefore provide a more direct indicator of the cytoplasm pH,. c. Uptake of9-Arninoacridine. An alternative to measurement of pH, with radioactive weak bases involves measurement of fluorescence quenching of 9aminoacridine. 9-Aminoacridine is a weak base and accumulates by diffusion of the unprotonated amine (Schuldiner et al., 1972; Christen et al., 1982). It has the property that its fluorescence is highly quenched upon uptake by sperm. Measurement of fluorescence quenching is equivalent to following disappearance of the 9-aminoacridine from the seawater as it is accumulated. Up to 80% of the dye may be taken up, yielding Ar values of 2200 or larger (Schackmann et al., 198I ; Christen er al., 1982). If a pH, value were determined directly from Eq. ( l ) , a pH, of -4.7 would be calculated, in complete disagreement with values for other weak bases. However, a careful study revealed that most acridine uptake occurs as a result of the intracellular binding of the dye following accumulation as weak base (Christen et al., 1982). That is, even if ApH is completely collapsed with detergents or ionophores, substantial uptake remains. The binding properties of the sperm for 9-aminoacridine were studied and it was found that at an initial external concentration of 1-2 pM, intracellular binding was far from saturation. As explained in detail in the supplement to the paper by Christen et al. (1982), this allows for correction of the binding in a simple manner to give the following equation:
64
ROBERT SCHACKMANN
where Q is fraction of fluorescence quenched (fraction of 9-aminoacridine taken up) and Ql is the fraction quenched in the presence of either detergent or ionophores to collapse ApH across the plasma membrane. The unexpected advantage of this method of pHi measurement is that intracellular water space need not be measured. With this correction pH, values determined by uptake of 9aminoacridine agree well with those determined by uptake of [ 14C]diethylamine or with 31P-NMR(Christen er al., 1982, 1983b). 2. pH, MEASUREMENTS BY 31P-NMRAND
WITH
CARBOXYFLUORESCEIN
Two measurements of sea urchin sperm pHi by 31P-NMRhave been reported (Christen et al., 1983b; Johnson er al., 1983). Measurement of the chemical shift of the 31Piis directly related to pHi (for review, see Nuccitelli and Deamer, 1982). The chemical shift of Pi varies with the degree of protonation of Pi and is a good indicator of pHi in the vicinity of the pK of Pi. Measurement of pHi therefore necessitates a pH calibration of the Pi chemical shift within the sperm. Christen and colleagues (1983b) approached this problem by collapsing the ApH with the ionophores nigericin and monensin so that pHi = pH,. Varying pHe and recording the Pi chemical shift allows for a calibration within the intracellular environment. Values determined by 31P-NMRin sperm are in excellent agreement with those determined by uptake of diethylamine (Christen et al., 1983b). For example, at pHe8 for sperm in artificial seawater, the pH, was 7.4 by 31PNMR, 7.3 by [ 14C]diethylamineaccumulation, and 7.4 by 9-aminoacridine uptake (Christen et al., 1982). And for quiescent sperm in Na+-free seawater, pH, was 6.8 by 31P-NMR and 6.9 by [14C]diethylamine uptake. A limitation of NMR is the requirement for high cell concentration. A 10% sperm suspension was necessary to yield a satisfactory spectrum in a reasonable amount of time (minutes). This precludes time-dependent resolution of pH, changes which are complete in seconds. However, 31P-NMRdoes provide the only reasonable method of analyzing the pHi in dry sperm before dilution. A pH, value of -7 was obtained, which is sufficiently low to explain sperm immotility in semen (Johnson er af., 1983; Christen er al., 1983b). Carboxyfluorescein and other optical pH probes have yet to be used as pHi probes for sea urchin sperm. A detailed report of carboxyfluorescein use for measurement of pHi in bovine sperm has been published by Babcock (1983) and the interested reader is referred to this article. It is mentioned here because intracellular optical probes provide the possibility of real-time resolution of the pHi changes important to sperm activation. For sea urchin sperm, measurement of pHi by following weak base uptake is
4.
65
ION MEASUREMENTS IN SEA URCHIN SPERM
the most direct method and can be performed without elaborate equipment. Uptake reflects the pH, of the apparent “cytoplasm” since it is in agreement with values determined by 31P-NMR and the bases will not accumulate into the mitochondria. It remains to be seen whether weak base uptake will be useful for evaluation of pH, in sperm of other species. The large acrosomal region of mammalian sperm has led Meizel and Deamer (1978) to propose that 9-aminoacridine uptake measures the intraacrosomal pH in hamster sperm.
IV. Measurement of Cation Uptake In the previous section pH, was discussed as it is an important regulatory component of sperm motility and the acrosome reaction. pH, changes in sea urchin sperm correlate with or are linked with other cation movements as well. In particular, a Na / H exchange is a component of the sperm plasma membrane that can either raise or lower the pH, (Lee, 1984a,b). Furthermore, induction of the acrosome reaction results in multiple changes in the sperm cation composition (Schackmann et al., 1981; Cantino ef al., 1983). Na+ and Ca2+ enter the sperm and K (as well as H +) exit. Of these Ca2 entry is a crucial requirement for the acrosome reaction regardless of the method used to initiate it. +
+
+
A.
+
Measurement of Ca*+ 1. 4sCa2+ UFTAKE
The most frequently used method for measurement of Ca2+ entry into sperm consists of following 45Ca2+ accumulation. The basic methodology is the same as that for monitoring uptake of weak bases (Section III,B,l ,a). Isotopic 4sCa2+ is added to the seawater and incubated with the sperm under the experimental conditions to be tested. Termination of uptake is effected by rapid separation of the sperm from the medium by either centrifugation or filtration. Most experiments have been performed by filtration (Schackmann et al., 1978; Kopf et al., 1984; Kopf and Garbers, 1980). I have found that centrifugation of sperm through silicone fluid works equally well, although background 45CaZ uptake is higher (-4 nmol/108 sperm) than for filtration (-1 nmol/108 sperm). Whether this represents rapid Ca2+ uptake, CaZ+ binding to the surface, or both, is not known. When the acrosome reaction is triggered, increased 45Ca2+ uptake is initiated and may reach -20 nmol/108 sperm (Schackmann et al., 1978). Such an increase is easily resolved by either filtration or centrifugation. Stimulation of 4sCa2 entry indicates that enhanced Ca2 influx occurs but does +
+
+
66
ROBERT SCHACKMANN
not provide direct evidence that net Ca2+ uptake results. In order to show that a net increase occurred, atomic absorption spectroscopy and X-ray microanalysis were performed. I will not deal with the methodology directly but refer the reader to work by Cantino and colleagues (1983). The results obtained by either method show that net Ca2+ uptake does correspond to 45Ca2+ uptake. X-Ray microanalysis also allowed for localization of the Ca2 uptake to the mitochondria as predicted from inhibitor studies (Schackmann and Shapiro, 1981). The slow, long-term accumulation of Ca2 for -30 minutes after the acrosome reaction is complete represents a complex function of Ca2 entry through the plasma membrane and accumulation by the mitochondrion. An additional factor limiting the usefulness of 45Ca2+ uptake is the level of resolution attained. With a background level of 1-4 nmol/108 sperm, it is extremely difficult to resolve increases of even 0.1 nmol/ lo8 sperm as were found when the acrosome reaction was initiated in the presence of drugs to inhibit mitochondria1 Ca2+ uptake (Schackmann and Shapiro, 1981). Yet a change of this magnitude could still represent an extremely large change in [Ca2+Ii.A need for measurement of changes in free [Ca2+Iiis clearly indicated. +
+
+
2.
MEASUREMENT OF [Ca2+Ii
Tsien et al. (1982) and colleagues (Grynkiewicz et al., 1985) have developed several Ca2+ indicator dyes which show altered fluorescence upon binding of Ca2 . These dyes can be loaded into cells as uncharged molecules if the carboxyl groups involved in binding Ca2+ are first esterified. The ester groups are hydrolyzed within cells to generate the native Ca2+ indicators. These dyes, “Quin2” (Tsien er al., 19821, “fura-2,” and “indo-1” (Grynkiewicz et al., 1985), have dissociation constants of 100 nM and are sufficiently insensitive to [Mg2+] and [H+]to allow for measurement of [Ca2+] within living cells. As highly charged anions, they do not readily leak from cells. These dyes have only recently begun to be applied to sperm physiology. In an experiment described in Table I, nigericin, a monovalent cation inonophore that triggers the acrosome reaction and stimulates 45Ca2+ uptake (Schackmann et al., 1978), was found to increase Quin2 fluorescence. The data indicate that [Ca2 ] increases approximately 8-fold within a minute following addition of the nigericin. Further application of these dyes to sperm physiology will allow for a more complete understanding of [Ca2+ I i changes important to sperm activation. +
-
+
B. Measurement of Na+ and K + The simplest method for measurement of Na+ or K + uptake is to follow 22Na+ or 42K fluxes. 42K has a 12-hour half-life and 86Rb has been used as a K + substitute (Schackmann et al., 1978). Recent attention has focused on +
+
+
4.
67
ION MEASUREMENTS IN SEA URCHIN SPERM
TABLE I NlGERlClN INCREASES
Conditions ASW. 5 mM Ca2+ + nigericin (60 phf) ASW, 0 mM Ca2+ + nigericin (60 phf) ASW, 5 mM Ca2+ + ionomycin (30 phf) + ionomycin + 3 mM Mn2+
[Ca2+]t'
Fluorescence (arbitrary units)
(Ca2 Ii (units of K d )
43 91 41 40
0.30 2.3 0.27 0.25
I22 0
-
+
Sperm (10% dry sperm by volume) of the sea urchin, S. purpuratus, were incubated 5 hours in calcium-free seawater, pH 6.8, with I mM EGTA and 50 phf Quin2-acetoxymethyl ester (Tsien et a / ., 1982). After washing the sperm and resuspending the pellet as 20% dry sperm, 100 pl was added to I .9 ml ASW at the indicated [Ca2+]. Ionophores were followed by I-minute incubations prior to fluorescence measurements. [Ca2+] was calculated as ( F F,,,/F,,,, - F)Kd (Tsien et a / ., 1982). where F = fluorescence and F,,, = the fluorescence with 5 mM Ca2+ and ionomycin. FIni,= 0.16 AF where A F is between F,,, and the F with ionomycin + Mn2+ to quench Quin2 fluorescence (Hesketh el a / . . 1983). Kd is the dissociation constant for the Quin2Ca2f complex within the sperm.
22Na+ movements because Na+ / H + exchange has been linked to pH, changes associated with motility activation (Lee, 1984a; Hansbrough and Garbers, 1981). Uptake of 22Na+ is complete within seconds when motility and respiration are stimulated by Na+ addition to sperm in Na+-free seawater (Lee, 1984a; Schackmann, unpublished data). 22Na+ entry is rapid when the acrosome reaction (Schackmann and Shapiro, 1981) is triggered and is sufficiently rapid in intact sea urchin sperm tails (Lee, 1984a,b) to preclude a kinetic analysis. These studies of 22Na uptake were performed by filtration on glass fiber filters (Whatman GF/C). As noted in Section IV,A, 1, for 45Ca2 uptake, both centrifugation and filtration gave comparable results. However, centrifugation of the sperm through silicone oil gives values of 22Na uptake approximately two-fold higher than by filtration (Cantino et af., 1983) and these values are in much closer agreement to those determined by X-ray microanalysis. Presumably, when sperm are filtered and washed, some 22Na+ exits rapidly. The time taken for 22Na+ to equilibrate across the sperm membranes is about the same magnitude as the time necessary for washing the filters and could lead to underestimates for Na uptake. Such apparent differences demand careful attention when quantifying Na+ uptake. The recently described N a + / H + exchange in isolated tails from sea urchin sperm is thought to be electrically neutral (Lee, 1984b) on the +
+
+
+
68
ROBERT SCHACKMANN
basis of an apparent 1:1 stoichiometry. Since different techniques yield different values for Na uptake, arguments for electroneutrality based on filtration measurements alone are unwarranted without careful substantiation. Even larger differences between data acquired by filtration and centrifugation have been registered for K uptake (Cantino et al., 1983). Again, centrifugation yields values in closer agreement to those determined by atomic absorption or Xray microanalysis. An additional difficulty exists with 22Na+ measurements. Evaluation of ion uptake is difficult if the ion is not concentrated within the sperm; that is if Ar 5 1. This means that measurement of K is quite simple since the Ar, + = -20 but for sperm in artificial seawater ATNa+= -0.3. The amount of Na trapped in the extracellular space is at least 50% of the total amount pelleted with the sperm. At normal levels of seawater Na+ both methods, filtration and centrifugation, are experimentally difficult to use as accurate quantitative measurements for Na+ uptake. +
+
+
+
V . Measurement of Membrane Potentials in Sperm The small size of sea urchin sperm has precluded attempts at measurement of membrane potentials with conventional microelectrodes. Measurements on intact sperm have relied on uptake of the diffusable lipophilic ions [ 14C]thiocyanateand [3H]tetraphenylphosphonium+(TPP+) (Schackmann et al., 1981, 1984). This method was designed to measure membrane potentials in bacteria and in membrane vesicles where electrode technology was not applicable (Lichtshtein et al., 1979; Rottenberg, 1979). If a negative membrane potential exists, the TPP+ cation will accumulate and the thiocyanate anion will be excluded (Ar < 1). We verified this for sperm of the sea urchin Strongylocentrotus purpuratus. Additionally, [3H]TPP+ uptake was decreased by increasing seawater [K+ 1, an event that is consistent with the depolarization of the plasma membrane potential by elevated extracellular [K 1. Conversely, [ I4C]thiocyanate uptake increased with increasing seawater [K+]. Qualitatively these data imply that the sea urchin sperm plasma membrane potential is similar to that of many other cells. Quantitative evaluation of the potentials with these membrane potential probes is more difficult, particularly for TPP+ , which largely accumulates into the mitochondrion (Schackmann et a f . , 1984) by virtue of the large negative mitochondrial potential. We were able to explore the contribution of mitochondria1 potential in intact sperm using [14C]thiocyanate ([ I4C]SCN) to measure the plasma membrane potential and using [3H]TPP to measure a composite potential that is dependent upon the two negative potentials acting in series. Additionally, by making the plasma membrane permeant with digitonin, we were able +
+
4.
ION MEASUREMENTS IN SEA URCHIN SPERM
69
to generate with an exogenous substrate a mitochondria1potential consistent with our in vivo measurements (- 155 mV). If corrections are made for the mitochondrial accumulation of TPP+ , both TPP+ and SCN yield values of the plasma membrane potential of -30 mV for sperm in artificial seawater. [14C]SCN uptake in principle provides a more direct measurement of the plasma membrane potential; but since Ar < 1, the error in these measurements is large. Measurements of [3H]TPP+ and ['4C]SCN uptake have been made principally by centrifugation through silicone oil (Schackmann et al., 1984). TPP+ was found to have limited solubility in the silicone oil. Diffusion from the sperm pellet was not substantial within 24 hours, but diffusion from the supernate into the silicone oil layer was found to erroneously enhance the value for Ar (supernatant counts decreased). Samples should, therefore, be processed immediately following an experiment. Although we have used the lipophilic ions to provide the first values for membrane potentials in sea urchin sperm, the methods have serious limitations. The kinetics of TPP+ uptake into sperm are extremely slow, thus only static, long-term charges in potential can be observed. It has proved possible to measure a depolarization of -30 mV when the acrosome reaction is induced. But if transient changes occurred prior to the net depolarization, we would be unable to detect them. And like uptake of weak bases, the time-course for changes in uptake does not reflect the time-course for changes in A+, but reflects a complex function of the potentials and sperm permeability to the probe molecules. Recent advances in patch-clamping small sections of cell membranes (Neher et al., 1978) should ultimately allow more exact, rapid measurements of sperm membrane potentials. In this regard, LiCvano and colleagues (1985) have only recently reported making lipid bilayers of sea urchin sperm membranes on a patch electrode. They found K + channels which could be blocked by tetraethylammonium. This potentially represents an exciting new method of analyzing ion movements through sperm membranes.
-
VI. Conclusion I have briefly attempted to provide the reader interested in measuring ionic changes in sperm with a perspective on how the measurements have been and should be made and the potential limitations inherent with existing techniques. Simple ionic manipulation, regulation of pH,, and [Ca2+] are of fundamental importance to sperm physiology. Investigations of ionic changes associated with activation of sea urchin sperm have allowed us to begin to define how these important events are initiated and lead to activation of development. Whether these changes are unique to echinoid sperm or are general characteristics of
70
ROBERT SCHACKMANN
sperm remains to be shown. Similar ionic changes, increases in pHi, and [Ca2+Ii are known to attend to a host of cell activation events throughout the animal kingdom. The apparent similarity between echinoid sperm activation and activation of other eukaryotic cells raises additional interest in the study of echinoid sperm and the ionic changes important to their activation. ACKNOWLEDGMENTS I wish to thank Merry Peters for typing this manuscript. This work was supported by Grant HD 18670 from the National Institutes of Health.
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