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Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2
Cell Calcium (1988) 9, 45-56 C’ Longman Group UK Ltd 1988
Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2 A.M. SIMPSON and LG. WHITE
Department of Veterinary Physiology, University of Sydney, Australia
Abstract - The highly selective fluorescent Ca2+ indicator ‘quin 2’ has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, ‘quin 2/AM’, which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean + S.E.M.) indicated a normal cytoptasmic free-calcium concentration, [Ca2+]i, of 193nM f 0.2 (n= 10) for ejaculated ram sperm, 175nM 5 3.9 (n= 10) for cauda epididymal boar sperm and 105nM ‘_ 10 (n= 10) for the caput sperm. After cold shock ejaculated mm and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium aride, propranolol and caffeine did not affect the [Ca2+]i of mm and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rote none and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrtal calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4DNP and CCCP had no effect on [Ca2+]i. Addftfon of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.
The physiological actions of calcium as a trigger and second messenger for co-ordinating events in spermatozoa, e.g. capacitation and the acrosome reaction [l-3], are presumably dependent on the regulation of a calcium gradient across the plasma membrane. A low intracellular calcium concentration relative to that in the seminal plasma is probably maintained through the operation of several mechanisms including the ATP-dependent calcium pump or the (Ca2+ + Mg’+)-ATPase of the
plasma membrane [4, 51 which has been partially characterised for ram and bull spermatozoa [6-9J. When sperm of the ram, boar and several other species are cold shocked by rapidly cooling to O”C, motility and metabolic activity are irreversibly decreased and the plasma membrane disrupted [lO191. Atomic absorption speclrometric analysis suggests that these changes may be accompanied by an increase in the calcium content of the sperm [14, 161 and a rapid influx of “Ca2+ has been demon45
46
strated on cold shocking ram and boar sperm 117, 191. However, apart from our preliminary communication 1201and a recently published study on human sperm 1211,there appears to be no information on the actual levels of free-calcium in the cytoplasm of mammalian spermatozoa. Estimation of the presumably critical cytoplasmic intracellular free-calcium concentration, [Ca2+/i, in a!! cell types is technically demanding and often involves measuring calcium fluxes by artificially increasing the pool with ionophores 1221, inserting ion-selective microelectrodes or injecting calcium indicators or buffers into single cells 1231. A few types of small cells can be loaded with buffers or indicators during controlled lysis [24-261but such procedures perturb membrane integrity and soluble cytoplasmic components. The present study utilises a new highly selective fluorescent calcium indicator ‘quin 2’ [27-301 to measure the cytoplasmic free-calcium concentration of ram and boar sperm after manipulation by compounds that affect calcium flux. Quin 2 is a tetracarboxylic acid which shows high affinity for calcium and very low affinity for magnesium and hydrogen ions. It can be non-disruptively loaded into cells by incubation with its acetoxymethyl ester ‘quin 2-AM’ which permeates cell membranes [29] and is hydrolysed in the cytoplasm, trapping the impermeant quin 2. In experiments with lymphocytes [29, 301 the intracellular quin 2 appeared lo be free in cytoplasm, not bound to membranes and not sequestered inside organelles such as mitochondria. In view of the pivotal role calcium plays in regulating many sperm functions 131 and its possible link with c-AMP, this paper also reports on the [Ca” +]i of ram and boar sperm after addition of dibutyry! c-AMP and the phosphodiesterase inhibitors, theophylline and caffeine which arc known to stimulate motility 131-331and to inhibit the uptake of “Ca2+ in ram and boar sperm [341. Other aspects of calcium metabolism were investigated by adding rotenone and antimycin A which prevent the stimulatory effects of calcium on phosphate accumulation by bull sperm 1351and decrease calcium and oxygen uptake in ram and boar sperm [34]. Lanthanum, an inhibitor of divalent cation uptake in rat liver mitochondria [36-381,
CEL,L CALCIUM
and carbony! cyanide-In-chlorophenylhydrazone (C’CCP) and 2,4-dinitropheno! (2,4-DNP), uncouplers of oxidative phosphorylation in mitochondria, were also tested along with sodium azide and propanolo! which inhibit the motility, but not the calcium uptake of human sperm [39]. In addition attempts were made to manipulate (Ca2+]i with the divalcnt cation ionophore A23L87, which initiates calcium influx in bull, boar, human and ram sperm (34,40,41]. Experiments were also undertaken with the polyene antibiotic blipin, a membrane disrupting agent which has been shown to release calcium accumulated in the presence of the ionophore [34,40].
Materials and Methods Collectiorl and processing of smell
Semen was collected from rams by electrical stimulation [42J and from the cauda epididymis of boars within three hours of slaughter by the method of Lasley & Bogart 1431. To obtain caput epididyma! sperm, the distal caput portion of the boar epididymis was removed and cleaned of blood vessels and connective tissue. Longitudinal incisions, approximately 5mm apart, were made and epididyma! semen extracted by gently swirling each section with medium containing 145mM NaC!, 5mM KC!, 1mM NazHP04, 1mM CaC!?, 0.5mM MgSQ, 5mM glucose and 1OmM HEPES titrated with NaOH to pH 7.4 (unless otherwise stated) at 37°C 1291. Caput semen from each section was pooled and !i!tered through 8 ply gauze (Handy) to rid the extract of tissue debris. Suspensions of the ejaculated ram and epididyma! boar spermatozoa were diluted in 5 volumes of the collection medium and centrifuged for 12 min at 500g and 2lYC. The supernatant was withdrawn, the sperm resuspended in medium and the washing procedure was repeated. The spermatozoa were !ina!!y resuspended in medium to give a concenlration of 1 x 10’ sperm/m!. Spermatozoa were counted in a haemocytometer. Motility of the sperm was estimated by light microscopy on a scale of 0 to 4 [44]. Washed spermatozoa were cold shocked by slowly pipetting
QUIN 1 AND CYTOPLASMIC
FREE CALCIUM
IN SPERMATOZOA
samples held at 25°C into glass vials maintained at 0°C in an ice bath. After 10 min all samples were brought to 30°C.
Calcium-magnesium buffers for calibration [29] were made up with Ca-Mg-EGTA at concenlrations calculated on the basis of apparent dissociation constants for Ca-EGTA and Mg-EGTA of 214nM and 8.96nM respectively at pH 7.05, 37°C (11SmM KCI, 20mM NaCl, 1OmM 4-morpholinepropanesulfonate (MOPS), 2.22mM EGTA and 1.22mM MgClz, titrated with KOH to pH 7.05 at 37°C). Fluorescence measurements were undertaken in the solution used for collection of semen. Additions of calcium were made from 1.0 or 0.2M CaCl2 stock and EGTA, pH was adjusted with 1M HCl or LM Tris base. All standard solutions were filtered through 0.45t.r.m Millipore filters prior to use and all glassware was acid washed and rinsed in a concentrated EGTA solution lo remove any contaminating calcium. Stock solutions of quin 2/AM were made up to 5OmM in dry dimethylsulphoxide (DMSO) and kept dessicated at -20°C between use. The linal concentration of DMSO from the quin 2/AM stock did not exceed 0.5% (vol/vol). A23187 was stored as a 1OmM solution in dimethylformamide/ethanol(3/1) al -20°C.
47
perature for 4 hours with no loss of quin 2 activity. For measurement of fluorescence. lml of the stock suspension was brielly centrifuged at 5OOOgand the sperm resuspended in 2ml of the fluorescent medium and transferred to the cuvette. This final step minimised the carry-over of external quin 2. The resulting quin 2 loading was approximately 3mM; a high intracellular quin 2 concentration will act as a calcium buffer and so perturb the (Ca”]i. All experiments involving quin 2 were replicated on sperm from at leas1 3 ejaculates.
Fluorescence was measured with a Perkin-Elmer LS-5 spectrofluorimeter. Standard monochromator settings were 339nm excitation with 5nm slits, and 492nm emission with 1Onm slits; 339nm was chosen as at this excitation wavelength the calcium and magnesium complexes of yuin 2 adsorb equally - and longer wavelengths would decrease the sensitivity to calcium binding. Samples were contained in lcm square quartz cuvettes thermostatically maintained at 37°C. Interruptions of the traces presented in the results show where the sample compartment was opened either to add reagents or stir suspensions with a plastic pipette. Statistical arta!vsi.s Differences between mean treatment values were assessed by t-tests.
and control
Cltertticals Sperm were loaded with quin 2 by a modificaton of the procedure described for lymphocytes by Tsein et al. 129) in the medium used for the collection and washing of sperm. Quin 2/AM (50uM) was added to the sperm suspension (10’ sperm/ml) and shaken for 20 min at 3PC in the dark. The suspension was then diluted with an equal volume of the same medium, incubated for a further 40 min (ram sperm) or 60 min (boar sperm), centrifuged at 1OOOgfor 4 min and the sperm resuspended in fresh medium. This suspension could be kept in the dark at room tem-
Quin 2 and quin 2/AM were obtained from Calbiochem, La Jolla, California, USA; A23187 and anlimycin A from Boehringer Mannheim, Mannhcim, West Germany; caffeine, propranolol, sodium azide, rotenone, dibutyryl c-AMP, theophylline and carbonyl cyanide-ln-chlorophenylhydrazone (CCCP) from Sigma Chemical Co., St Louis, Missouri, USA; 2,4_dinitrophenol (2,4DNP) from Ajax Chemicals, Sydney, Australla. Filipin was a gift from Upjohn International Inc., Kalamazoo, Michigan, USA. All other chemicals were of analytical reagent grade.
48
CELL
CALCIUM
Results Spectra of qttin 2 ii; presence of calcium
Preliminary experiments were undertaken to check that the quin 2 method of Tsein et al. [29] was suitable for measuring intracellular calcium in sperm. The emission spectra of quin 2 were observed in the presence of free Ca2+ ranging from a concentration of ‘0’ (< 1nM) to a saturation level of 10Ot~M. The solutions were Ca*+-EGTA buffers with concentrations of free cations chosen to mimic the intracellular environment. With excitation at 339nm, the intensity of fluorescence increased about six-Cold over a range of [Ca’+] from 0 to lOOp,M although the wavelength of the emission peak remained nearly constant at 492nm. The fluorescence response was nearly maximal for calcium concentrations above lt~,M. The dependence of fluorescence at 492nm on free-calcium was checked for this system and the results conformed to the theoretical behaviour of a simple 1 dye: 1 Ca” + binding with an effective dissociation constant of 115nM. The absence of free Mg*+ resulted in an effective dissociation constant of 60nM as found by Tsein et al. [29].
%
s
2 P
I’.
;
~~~_
J1-Pom’n
i
_/
400
I
450
500
1 550
I 800
mm
E’ig. 2
Shifts of emission spectra during quin 2/AM
uptake
and hydrolysis in ram sperm. Excitation 339n?;The initial fluorescen e of quin 2/AM is shown in ImM Ca solution and s then 10 speml were added. Dashed curves show the suspension spectra at 20 and 60 min. The curve peaking at 492nm was obtained after washing the sperm. resuspending in the same volume of fresh medium then releasing the trapped dye with Triton X-100. The bottom trace ‘sperm alone’ shows the autofluorescence measured from unloaded cells at the same density
The emission spectra
of 20uM quin 2 at
‘O’Ca* + ( c lnM), 2OOnM Ca*+ and 1mM Ca*+
are shown in Figure 1 at pH 6.75, 7.05, 7.50 and 8.00. The [Ca*+] was altered by appropriate additions of Ca-EGTA, CaClz and K-MOPS while carefully adjusting the pH. The spectra at ‘0’ Ca” ’ were unaffected by pH change but at 1OOnMCa*+, lowering the pH from 7.05 to 6.75 inhibited fluorescence. However, raising the pH to 7.5 and 8.0 had little effect on the emission spectra. It is important to note that the effect of pH on the affinity of quin 2 for calcium is difficult to study because EGTA is much more pH-dependent than quin 2, thus in latter experiments in which pH was altered very careful adjustments were necessary. Loading of quirt 2 into spem 400
410
500
IS0
000
of quin 2/AM in sperm was easily monitored by the gradual shift in the emission spectrum from that of the ester which peaks at 430nm, to that of quin 2 itself peaking at 492nm. This is shown in Figure 2 for an experiment on ram sperm in which
Hydrolysis
Fig. 1 (cInM
Ey$sion spectra $ri: 20f.tM quin 2 at ‘0’ Ca2+ Ca ), IOOnM Cy: and 1mM Ca*+ at pH 6.75. ] and pIi were altered by appro-EGTA.
careful adjustment of pH
CaC12 and K+-MOPS
with
QUIN 2 AND CYTOPLASMIC
FREE CALCIUM
49
IN SPERMATOZOA
Table 1 Motility scores (+ S.E.M.) for three observations of ejaculated ram and cauda boar sperm in the presence and absence of 3mM quin 2 Sperm
Addition
0
lhr
Ejaculated ram
Control Quin 2
3.6 2 0.3 2.6 2 0.3
3.4 -+ 0.3 3.2 f 0.2
3.4 + 0.3 3.2 + 0.1
3.0 2 0.1 2.9 ? 0.2
Cauda boar
Control Uuin 2
3.2 + 0.1 3.2 ? 0.1
3.2 + 0.2 3.2 f 0.1
3.0 -t 0.2 2.9 +- 0.1
3.0 f 0.2 2.8 ‘-r-0.1
5OpM quin 2lAM was added to 10’ sperm/ml; the dashed curves show the emission spectra at 20 and 50 min by which time the spectral shift was almost complete. A large proportion of the quin 2 was trapped inside the sperm, as demonstrated by centrifuging and resuspending the sperm in fresh medium to remove external quin 2. It took longer (approx. 80 min) to load boar epididymal sperm with quin 2 but the curves were similar to ram sperm (Fig. 2). Because the dye measures [Ca”+] by a change in fluorescence intensity, the signal depends on [Cal+] as well as the dye content of the suspension and in order to measure the [Ca’ + 1in sperm it was necessary to mix the dye with known levels of Ca’+ while keeping the dye content of the suspension constant. This was done at the end of each experiment by releasing the dye from sperm with Triton X-100. The medium contained 1mM Ca” so that maximum fluorescence (Fmax) was obtained after releasing the dye. [Ca”+] was then set at 300 and 1OOnM by adding EGTA and MgCl2. Minimum fluorescence (Fmin) was obtained by adjusting the calcium concentration to 1nM by addition of 2nM EGTA and enough Tris base to take the pH to >8.3. The autofluorescence of unloaded sperm was also taken into account and an appropriate correction made to Fmaxand Fmin. The sperm regenerated quin 2 with the correct Ca’+ affinity and it was sufficient in routine experiments to note the Fmax and Fmin values in the lysate and calculate the [Ca’+]i from intact cells by substitution in the following equation [29]: (Ca”+l = Kd (F- F,in)/(F,,,-
F)
21Ir
3111
Fluorescence observations in several ex eriments indicated a normal intracellular [Ca 2-F) ] of 193nM +- 0.2 (n = 10) for ejaculated ram sperm, 175nM -c 3.9 (n = 10) for cauda boar sperm and more variable levels of 1lOnM 5 10 (n = 10) for caput boar sperm. Table 1 shows that quin 2 was not toxic to ejaculated ram and cauda epididymal boar sperm over 3 h as gauged by motility. As the movement of caput boar sperm was restricted to slight twitching before and after addition of quin 2, no results are presented. It is evident from Table 1 that the motility of ram and boar sperm is well sustained in the saline medium employed. If ram ejaculated or boar cauda sperm were first cold shocked at 0°C they could not be loaded subsequently with quin 2/AM. Furthermore, if normal sperm were loaded with quin 2/AM, exposure to cold shock caused slow release of quin 2 into the medium. In contrast, cold shocked caput boar sperm could be easily loaded with quin 2 and had a [Ca”+]i of 117 + 8.8nM (mean + S.E.M.; n =4) which was similar to the 121 ? 6.9nM of control sperm. Effect of inhibitors, activators and swfactam
Figures 3 and 4 show the effect of metabolic inhibitors, activators and surfactants on the intracellular calcium levels of ejaculated ram, and cauda and caput boar sperm. The inhibitor sodium azide and the B-adrenergic antagonist, propranolol, which abolished the motility of sperm from both species, had no significant effect on the [Ca”]i of the sperm. Similarly the phosphodiesterase inhibitor caf-
50
CELL CALCIUM nY
1OmY
6mY
Na
Ca
2+
AZIDE _
196
_ -
206 192
CAFFLIWE
SmM
nM
DIBUTYRYL
Cm’+
1.2mMPROPI1ANOLOL r
,102
6mM I
THtDPHYLLlNC
CAMP
-1,)
2#Y CCCP
-220 -1e3
\ luM AWTIYYCIN
I
6 WIN
2.6mY
A
-106
L.)+
I
Fig. 3 Reproductions of spectrofluorimeter of ejaculated ram sperm
traces showing the effect of substances on the cytoplasmic free calcium concentration
feine which stimulates motility in boar caput sperm (341 produced no significant change in calcium levels of the sperm of either species. By contrast the other phosphodiesterase inhibitor, theophylline, and dibutyryl c-AMP produced a ~~dual but significant (P ~0.01) decrease in [Ca ]I in both ram and boar sperm. The decreases in [Ca*+]i (mean -c S.E.M., n=3 or 4) after 5 minutes exposure were: ram, 193 + 0.3 to 107 f 5.6 (theophylline), 193 + 0.4 to 91 f 0.9 (dibutyryl c-AMP); boar cauda, 175 f 4.1 to 98 f 8.1 (theo-
phylline), 174 f 3.8 to 119 f 8.1 (dibutyryl cAMP); boar caput, 113 + 1.2 to 54 + 3.1 (thcophylline), 118 f 2.1 to 56 f 7.8 (dibutyryl cAMP). The inhibitors rotenone and antimycin A, and the uncouplers of oxidative phosphorylation, CCCP and 2,4-DNP, produced a transient elevation of [Ca*+ Ii with a subsequent return to control levels or below. Addition of La3+ caused a rapid decrease (P < 0.05) in the [Ca*+]i of ram and boar sperm to a
WIN
2 AND CYTOPLASMIC
FREE CALCIUM
CAUDA
BOIR
IN SI’ERMATOZOA BOAR
CAPUT
SPERM
SPERM
ClY c.‘+ lomY
F
Ya
1
C.2+
AtlOf!
-1T6
l.¶mY
w 1
lnoPaANoLoL
l.tmM
-116
6mY
p
8mY
1
-110
CAFFEINE
A
T~LO?liVLLlN6
-110 IMY
-I?6
DIBUTVRYL
PROCRAWOLCL
6mY
CACCSINI
-1t6
5mY I
nN I(8
AZIDE
CAMP
--(‘O
THLOPHY
LLINL
,--!-+-L_ 1’:: SKY
DIBUTYRYL
CAMP
1’::
-i__ SUN
,6uN
ROTLWOWC
ANTIMVCIN
L
‘It6 SOYY
A
-
60
-
26
1.4-OWC -166 -176
L
\ 2.6mN
-
60
I..*+
2.6mN
1
L.‘+
-176 --t,____
Fig. 4 Reproductions of spectrofluorimeter tion of ejaculated boar spermatozoa
tracings
showing the effect of substances
Y7:
on the cytoplasmic
free-calcium
concentra-
52
CELL
EJACULATED
RAM SPERM
CAUDA nY
A.pH
Ca
BOAR
SPERM
II
nM Ca
CAPUT
21
BOAR
CALCIUM
SPERM nM Ca
r --
7.5
I+
450
i
I’ t
A23187
A23187
-3
-183
/ A23187
d -173
B.pH
Ii
\
8.0
-__i
-123
--7
-450
r+_._.w-
/
(
i
A23187
I
A23187
d1
-188
v
A231871
+..“.bJ
-180
kid -I
Fig.
5 Reproductions
calcium concentration
of spectrofluorimeter
-563
/
-440
-127
traces showing the effect of the ionophore A23187 (3.0l~,M) on the cytoplasmic free-
of ram and boar sperm incubated at (A) pH 7.5 and (B) pH 8.0
, 0
4
8
CONCENTRATION
12
16 OF
20 A23187
24
28
0
(#MI
Fig. 6 The effect of increasing concentrations of the ionophore A23187 on the cytoplasmic free-calcium concentration of ram sperm incubated at pH 8.0. Each point represents the mean f S.E:M. for 3 experiments
. 4
8
CONCENTRATION
Fig. 7
12
10 OF
20 A23187
The effect of increasing concentrations
24
28
(MM)
of the iono-
phore A23187 on the cytoplasmic free-calcium of cauda (triangles) and caput (squares) boar sperm incubated at pll 8.0. Bach point represents the mean k S.E.M. for 3 experiments
QUIN
2 AND
CYTOPLASMIC
FREE
CALCIUM
low level that was maintained throughout the exThe decreases in (Ca-+li after 5 periment. minutes exposure were: ram, 193 + 0.4 to 161 r 5.9; boar cauda, 175 f 3.7 to 102 + 6.2; boar caput, 114 + 3.1 to 71 + 16.7. Effect of the ionophore A23187
It was noted in an earlier study 1451that the ionophore-induced accumulation of “Ca”+ by ejaculated and cauda ram sperm increased under alkaline conditions but in boar sperm the calcium increase was less sensitive to changes in pH. Quin 2 measurements (y$. 5) indicated a large (P < 0.01) increase in [Ca ]i from 1%3 + 6.5 to 450 + 18.9nM (n = 4) in ram sperm at pH 8.0 on addition of 3.0t~,MA23187 compared to a smaller rise at pH 7.5. Once again raising the pH of the medium had little effect on the large increase in [Ca’+]i of boar cauda and caput sperm incubated with the ionophore. The effect of increasing concentrations of A23@7 on the [Ca”]i of ram and boar sperm at pH 8.0 are shown in Figures 6 and 7 respectively. The lower ionophore concentrations (0.33 to 3.0t~_M)rapidly increased (Ca”]i (P
RAM SPERM
53
IN SPERMATOZOA A.
EJACULATED
RAM
0.
CAUDA
SPERM
3YY
BOAR
C.
CAPUT
Fig. 8
BOAR
Reproductions
-200 *1
SPERM
of spectrofluorimeter
CAUDA BOAR SPERM
CAPUT BOAR SPERM
nM
ha’
nM Cm” FILIPIN
-176 +FILIClN
-_-
--._--
-273
--_I-
-420
-102 FILIPIN
r-i
A29
j
I’;:
k-
Fig. 9 (3.0&M)
traces showing
nophore to ram and boar sperm was apparently not dependent on oxidative metabolism of the mitochondria, as subsequent addition of the inhibitors CCCP and 2,4-DNP did not affect [Ca’+ Ii. In an attempt to detect any calcium stored in
-307
-193
-120
the effect on the cytoplasmic free-calcium concentration of adding (he ionophore A23187 to ram and boar sperm in calcium free medium
FILlPIN
-380
nM c.*+
A23187
L/-l_
nM CI~’
FILlPIN
SPBRM
Reproductions of spectrofluorimeter traces showing the effects of (A) filipin (0.2mM) plus filipin. on the cytoplasmic free-calcium concentration of ram and boar sperm
-
15
and (U) the ionophore
A23187
54
the mitochondria, or indeed any membrane-bound organelle, 3.0uM A23187 was added to ejaculated ram and epididymal boar sperm in CaZ+-free medium (Fig. 8) and resulted in a transient rise in [Ca’+ Ii. The polyene antibiotic filipin, forms a complex with cholesterol in biological membranes [46, 47) while leaving the mitochondria intact [40, 481. It increased (P < 0.01) [Ca’+Ji in ejaculated ram, and cauda and caput boar sperm (Fig. 9). However, 0.2mM filipin released calcium from ram and boar sperm previously exposed to ionophore which had increased [Ca’+]i. In the presence of the ionophore, the cytoplasmic free-calcium concentration was not affected by theophylline, rotenone or antimycin A, all of which altered the [Ca’+[i of control sperm. However, addition of La3+ to ionophore treated ejaculated ram and cauda boar sperm decreased (P ~0.01) [Ca”+ Ji from 420 f 9.lnM to 210 f 6.4nM and from 530 f 8.lnM to 170 + 13.lnM. These values did not differ significantly from those recorded for control sperm.
Discussion Quin 2 can be easily loaded into ram and boar spermatozoa as the acetoxymethyl ester which then hydrolyses. Loadings of several mM are not toxic to these spermatozoa as judged by motility scores and it has proved a suitable indicator of [Ca”+ Ji. The values obtained are similar to those for human sperm [21]. It has been shown that cold shocking ejaculated ram [19] or cauda boar sperm [34] causes damage to the plasma membranes and to the acrosome, and this structural damage would account for the failure of shocked ejaculated ram and cauda boar sperm, to retain quin 2. In contrast, caput boar sperm do not suffer the same structural damage on cold shocking to 0°C (341 and can therefore be readily loaded with quin 2 and have an internal calcium concentration similar to conlrol sperm. The concentration of inhibitors and pharmacologically active substances tested in these experiments are within the range required lo elicit their physiological effects [49]. However, it should be
CELL CALCIUM
remembered that in media containing glucose, spcrmalozoa can probably maintain their ATP by glycolysis and respiratory inhibitors may therefore, under these conditions, have little effect on the ATP concentration in the sperm or the Ca” ’ /Mg2 + ATPase of the plasma membrane. The metabolic inhibitor, sodium azide, and the surface active a ent, propranolol severely inhibited the uptake of 49Ca”+ by ram and boar sperm 134). However, the quin 2 fluorescence measurements suggest there is no change in [Ca2+]i. Although caffeine had no effect on the [Ca2+li of ram and boar sperm, theophylline and dibutyryl c-AMP gradually reduced calcium levels in a comparable fashion lo the inhibition of 45Ca2+ uptake (491. These results support the proposal of Peterson et al. [41] that theophylline and dibutyryl cAMP may act on the plasma membrane calcium pump through an interaction with c-AMP to reduce internal calcium levels in the sperm. As large increases in internal calcium are detrimental to the sperm, this may be a mechanism by which cyclic nucleotides maintain active motility. The transient elevation of sperm [Ca2+]i on adding the inhibitors, rotenone and antimycin A, and the uncouplers, CCCP and 2,4-DNP, was most likely the result of release of mitochondrial calcium stores. The return lo control levels or below was probably due to the extrusion of released calcium via the plasma membrane calcium pump. The addition of La3+ resulted in slow leaching out of internal calcium, presumably by disruption of normal calcium transport. The results obtained on adding low concentrations of the calcium ionophore A23187 to sperm were as expected 134, 40, 411 and its action of increasing [Ca’+]i was highly pH dependent in ram sperm in contrast to boar sperm. Quin 2 could not be used to monitor the rise in [Ca2+]i in the presence of high concentrations of the ionophore due to saturation of the dye. Apparently, the large increase of [Ca’+]i in response to A23187 was not dependent on the oxidative metabolism of the mitoc.hondria as subsequent addition of the uncouplers, CCCP and 2,4-DNP, had no effect on sperm [Ca’+]i in either species. The increase in the [Ca2+]i of ram and boar sperm induced by filipin is in agreement with other studies based on
QUIN
2 AND
CYTGPLASMIC
FREE
CALCIUM
55
IN SPERMATOZOA
murexide and 45Ca2+ uptake [34,40,41]. Addition of lilipin to ionophore treated ram and boar sperm, however, decreased ]Ca2+Ji. This would not be expected if there was gross disruption of the plasma membrane by filipin and similar results with bull sperm have been interpreted as evidence for lack of mitochondrial involvment in the calcium influx induced by A23187 1401. Addition of La3+ also reduced the ionophore-induced increase in [Ca’+]i, possibly by reaction with calcium carrier sites and disruption of the balance struck between the intcracting mitochondrial and plasma membrane calcium pumps. The increase in [Ca”+]i produced when A23187 was added to sperm in calcium-free medium quickly returned to control levels, most likely through extrusion by the plasma membrane calcium pump. The quin 2 measurements of cytoplasmic-free calcium in ram and boar sperm in the presence of A23187 support the suggestion of Babcock et al. [4O] that high concentrations of calcium and ionophore overwhelm both calcium membrane pumps and calcium enters a non-mitochondrial compartment of the sperm as a consequence of the equilibration of the ion across both mitochondrial and plasma membranes. The sensitivity of (Ca’+ ]i to filipin indicates that calcium binding to soluble intracellular components may also be involved 140, 411. The advantages of quin 2 as an indicator of [Ca2’]i in ram and boar sperm are that it can be non-disruptively loaded into the cells, it is nontoxic to the spermatozoa and it seems IO be as good or better than other reagents for quantifying calcium levels below lo-‘M. There is, however, a problem with autofluorescence in some sperm preparations and it is difficult to quantitate calcium levels above lt.~M as seen after addition of the ionophore. Similar problems have been encountered in measuring the [Ca2+]i of lymphocytes ]2Y]. Acknowledgements
The authors are indebted to Dr. 1. Kennedy for advice in setting up the assay, Dr. W. Jones for use of a spectrofluorimeter, Miss K. Murdoch for her invaluable technical assistance and to the Australian Wool Corporation for linancial support (I.G.W.) and a studentship (A.M.S.).
References 1. I ehninger
AI.. Car&Ii
E. Rossi CS. (1969)
linked ion movements in mitochondrial
Energy-
systems. Adv.
Bnzymol.. 29.259-320. 2. Berridge MJ. (1975) The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adv. Cyclic Nucleotide
Rcs.. 6. l-98.
3. Garbers DL. Kopf GS. (1980)
The regulation of spermato-
zoa by calcium and cyclic nucleotides. tide Res.. 13. 252-306.
Adv. Cyclic Nucleo-
4. Carafoli E. Crompton M. (1978) The regulation of intracellular calcium. In: Current Topics in Membranes and Transport (ed. F Bronner. A. Kieinzeller). 151-216. Academic Press. New York. 5. Lynch
I$
Vol. 10. pp
2t Cheung WY. (1979) Human erythrocyte Cg+ - ATPase: mechanism of stimulation hv Ca’
+ M,i‘ ’ Arch_Biochem. Biophys.. 194. 165-170. 6. Bradley MY. Forrester,IF (1980) A (Ca” + Mg2+)transport in the plasma memATPase and active Ca’ branes isolated from ram sperm flagella. 381-390. 7. Breitbart
Cell Calcium. I.
II Stern B. Rubinstein S. (1983)
port and Ca2’
Calcium trans-
-ATPase activity in ram spermatozoa
plas-
ma membrane vesicles. Biochim. Biophys. Acta. 728.349355. 8. Breitbart
H. Darshan R. Rubinstein S. (1984)
Evidence
for the presence of ATP-dependent calcium pump and ATPase activities in bull sperm head membranes. Biochem. Biophys. Res. Commun.. 122.479-484. 9. Breitbart H. Rubinstein S. (1983) Calcium transport by bull spermatozoa plasma membranes. Biochim. biophys. Acta. 732. J(y1-468. IO. Blackshaw AW. (1954) The prevention of temperature shock of bull and ram semen. Aust. J. Biol. Sci.. 7,573~582. II.
Blackshaw AW, Salishuty GW. (1957)
Factors influencing
melabolic activity of bull spermalozoa
in cold shock and its
prevention. J. Dairy Sci., 40. 1099-f 106. 12. Mann 1‘. Lutwak-Mann C. (195.5) Biochemical changes underlying the phenomenon Arch. Sci. Biol.. 39.578-588. 13. Wales RG. White IG. (1959) tozoa to temperature
of cold shock in spermatozoa. The susceptibility
shock. .I. Endocrinol..
of sperma-
19. 211-220.
14. Quinn PJ. White IG. (1966) The effect of cold shock and deep freezing on the concentration of major cations in spermatozoa. J. Reprod. Fertil.. IO. 263-270. 15. Quinn PJ. White IG, Cleland KW. (1969) Chemical and ultrastructural changes in ram spermatozoa after washing. cold shock and freezing. J. Rcprod. Perth.. 18.209-220. 16. Karagiannidis A. (1976) The distribution of calcium in bovine spemiatozoa
and seminal plasma in relation to cold
shock. J. Rcprod. I’ertil.. 46,83X1. I’.
Van Eerten M’IW.
Forrester
pects of ram sperm viability. Anim. Prod.. 40. 1.30-135.
I’]‘. (1980)
Biochemical as-
Proc. New Zealand Sot.
18. Simpson AM, White IG. (1986) Effect of cold shock and cooling rate on calcium uptake of ram spermatozoa. Anim. Reprod. Sci.. 12. 131-143. 19. Simpson AM. Swan MA. White IO. (1986) phatidylcholine in protecting tiamete Res.. 15.4356.
Action of phos-
rani sperm from cold shock.
56
CELL
20. Simpson AM. White ICi. (1985)
Measurement
and man-
36. Mela L. (1968)
Interactions lf+La3+
ipulation of cytoplasmic free calcium of ram and boar
drugs with mitochondrial
sperm.
Biochem. Biophys.. 123.286-293.
Proc. Aust. Sot. Repro. Biol, (25-28th August,
Adelaide).
49.
37. Mela L. (1969)
21. Stewart ID. Aitken
RJ. (1986)
Measurement
lar calcium in human spermatozoa.
of intracellu-
Gamete Res.. 15,57-
71.
pp 423432. Elsevier, Amsterdam. 23. Ashley CC. Campbell AK. (1979) Detection
and measure-
ment of free calcium ions in cells. Elsevier. Amsterdam. 24. Simons TJB. (1976) The preparation of human red cells ghosts containing calcium buffers. 25. Baker PF. Knight DE. (1978)
J. Physiol.. 256.209-244.
Calciumdependent
exocy-
tosis in bovine adrenal medullary cells with leaky plasma membranes. 26. Yi Ca%
JF. (1978)
meahility of human erythmcyte ghosts. Biophys. J.. 23. 403-471. 27. Tsein KY. (1981) A non-disruptive technique for loading calcium buffers and indicators into cells. Nature. 290, S27528. 28. Lew VL. Tsein RY. Miner C. (1982) Physiological (Ca2+ ]i level and pump-leak turnover in intact red cells measured using an incotporated Ca chelator. Nature. 298. 478-481. 29. Tsein RY. Pozzan T. Rink TJ. (1982)
trapped fluorescent indica-
30. Pozzan T, Arslan P. Tsein RY. RinkTJ.
tility and the transport and binding of divalent cations to the plasma membrane of human spermatozoa. Fertil. Steril.. 27. 1301-1307. 40. Babcock CF. First NL. I ardy HA. (1976) Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes. J. Biol. Chem., 251.3881-3886. 41. Peterson RN. Seyler D. Bondman 1). Freund M. (1979) The effect of theophylline and dibutytyl cyclic AMP on the
42. Blackshaw AW. (1954)
J. Rcprod. Fertil.. 55.385390.
A bipolar electrode for the produc-
tion of ejaculation in sheep. Aust. Vet. J.. 30. 249-250. 43. Lasley JP, Bogart R. (1944) A comparative study of the epididymal and ejaculated spermatozoa of the boar. J. Anim. Sri.. 3.360-370. 44. Emmens CW. (1947) The motility and viability of rabbit spermatozoa at different hydrogen-ion concentrations. J. Physiol.. 106.471-181. 45. Simpson AM, White IG. (1984)
Effects of pH on the cal-
(1982)
Anti-
cytoplasmic free calcium and capping in
tides on sperm respiration and motility.
Biochemistry.
IO.
1825-1831. 32. Garbem DI.. First NL. Gornran SK, Lardy I IA. (1973) The effects of cyclic nucleotide phosphodiesterase inhibitors on ejaculated porcine spermatozoan
Proc. Aust. Sot. Reprodl
Biol., (August, Melbourne). 46. De Kruijff B. Demcl RA. (1974)
Polyene antibiotic-sterol
interactions in membranes of A&&Q&malaidlawii cells and lecithin liposomes. Biochim. Biophys. Acta. 339,
B lymphocytes. J. Cell Biol.. 94. 335340. 31. Ciarbers DL. Lust WI). First NL, Lardy HA. (1971) Effects of phosphodiesterase inhihitors and cyclic nucleo-
metabolism.
Biol. Reprod., 8.599606. 33. Goh PT. (1982) Regulation of sperm function by calcium
57-70. 47. Bittman R. Chen WC. Andersen OR. (1974) Interaction of filipin 111and amphotericin B with lecithin-sterol vesicles and cellular membranes. Spectral and electron microscope studies. Biochemistry. 13. 1364-1373. 48. Morton BE. Lardy HA. (1967) Cellular oxidative phosphorylation. III. Measurement in chemically modified cells. Biochemistry. 6.57-61. 49. Simpson AM, White IG. (1987) Interrelationships between motility, c-AMP, respiration and calcium uptake of ram and boar sperm. Anim. Reprod. Sci.. In press.
PhD thesis. University of
Sydney. N.S.W.. Australia. 34. Simpson AM. (1986) Studies of calcium fluxes in ram and boar spermatozoa. PhD thesis. University of Sydney, N.S.W.. Australia. 35. Babcock DF, First NL. Lardy IIA. (1975) Transport mechanisms for succinate and phosphate localized in the plasma membrane of bovine spermatozoa. J. Biol. Chem.. 250, 64886495.
ions abolish the calcium re-
presence of ionophore A23187.
Calcium homeos-
tor. J. Cell Biol., 94.325334.
cyclic nucleotides and proteins.
Lanthanum
sponse of nerve terminals. Nature 299.410411. 39. Peterson RN. Freund M. (1976) Relationship between mo-
cium uptake and respiration of ram and boar sperm in the
tasis in intact lymphocytes: cytoplasmic free calcium moni-
immunoglobulin,
Effect of lanthanides and local an-
aesthetic drugs. Biochemistry. 8.2481-2486.
and human spermatozoa.
Changes of intracellular
as measured by arsenazo III in relation to the K per-
tored with a new intracellularly
Arch.
uptake of radioactive calrium and phosphate ions by boar
Nature, 276.620-622.
t DR. Hoffman
an 4 local anaesthetic
and Mn’ + uptake.
Inhibition and activation of calcium trans-
port in mitochondria. 38. Miledi R. (1971)
22. Lew VI,. Brown AM. (1979) Experimental control and assessment of free and bound calcium in the cytoplasm of intact mammalian red cells. In: Detection and measurement of free calcium in cells (ed. CC Ashley and AK Campbell).
Ca
CALCIUM
Please send reprint requests to: Dr. LG. White, Department of Veterinary Physiology. University of Sydney. 2006, N.S.W., Australia. Received: 19 May 1987 Accepted: 6 January 1988
Measurement and manipulation of cytoplasmic free calcium of ram and boar spermatozoa using quin 2 A.M. SIMPSON and LG. WHITE
Department of Veterinary Physiology, University of Sydney, Australia
Abstract - The highly selective fluorescent Ca2+ indicator ‘quin 2’ has been loaded into ram and boar spermatozoa as the acetoxymethyl ester, ‘quin 2/AM’, which is hydrolysed and trapped in the cytoplasm. Loadings of several mM were not toxic to spermatozoa as judged by motility. Fluorescence measurements (mean + S.E.M.) indicated a normal cytoptasmic free-calcium concentration, [Ca2+]i, of 193nM f 0.2 (n= 10) for ejaculated ram sperm, 175nM 5 3.9 (n= 10) for cauda epididymal boar sperm and 105nM ‘_ 10 (n= 10) for the caput sperm. After cold shock ejaculated mm and cauda epididymal boar sperm did not retain quin 2, due presumably to structural damage. However, cold shocked caput boar sperm could be readily loaded with quin 2 and had a [Ca2+]i similar to control sperm. Sodium aride, propranolol and caffeine did not affect the [Ca2+]i of mm and boar sperm, however theophylline, dibutyryl c-AMP and La3+ significantly reduced it. The inhibitors rote none and antimycin A, and the uncouplers 2,4-DNP and CCCP caused a transient elevation of [Ca2+]i, most likely resulting from release of mitochondrtal calcium. The increased [Ca2+]i following addition of the ionophore A23187, was highly pH dependent in ram spermatozoa and it was critical to increase the pH of the medium above 7.5; the increase in [Ca2+]i was apparently not dependent on the oxidative metabolism of the sperm as addition of the uncouplers 2,4DNP and CCCP had no effect on [Ca2+]i. Addftfon of filipin to ram and boar sperm resulted in a large increase in [Ca2+]i but addition of filipin to ionophore-treated sperm caused [Ca2+]i to fall well below control levels.
The physiological actions of calcium as a trigger and second messenger for co-ordinating events in spermatozoa, e.g. capacitation and the acrosome reaction [l-3], are presumably dependent on the regulation of a calcium gradient across the plasma membrane. A low intracellular calcium concentration relative to that in the seminal plasma is probably maintained through the operation of several mechanisms including the ATP-dependent calcium pump or the (Ca2+ + Mg’+)-ATPase of the
plasma membrane [4, 51 which has been partially characterised for ram and bull spermatozoa [6-9J. When sperm of the ram, boar and several other species are cold shocked by rapidly cooling to O”C, motility and metabolic activity are irreversibly decreased and the plasma membrane disrupted [lO191. Atomic absorption speclrometric analysis suggests that these changes may be accompanied by an increase in the calcium content of the sperm [14, 161 and a rapid influx of “Ca2+ has been demon45
46
strated on cold shocking ram and boar sperm 117, 191. However, apart from our preliminary communication 1201and a recently published study on human sperm 1211,there appears to be no information on the actual levels of free-calcium in the cytoplasm of mammalian spermatozoa. Estimation of the presumably critical cytoplasmic intracellular free-calcium concentration, [Ca2+/i, in a!! cell types is technically demanding and often involves measuring calcium fluxes by artificially increasing the pool with ionophores 1221, inserting ion-selective microelectrodes or injecting calcium indicators or buffers into single cells 1231. A few types of small cells can be loaded with buffers or indicators during controlled lysis [24-261but such procedures perturb membrane integrity and soluble cytoplasmic components. The present study utilises a new highly selective fluorescent calcium indicator ‘quin 2’ [27-301 to measure the cytoplasmic free-calcium concentration of ram and boar sperm after manipulation by compounds that affect calcium flux. Quin 2 is a tetracarboxylic acid which shows high affinity for calcium and very low affinity for magnesium and hydrogen ions. It can be non-disruptively loaded into cells by incubation with its acetoxymethyl ester ‘quin 2-AM’ which permeates cell membranes [29] and is hydrolysed in the cytoplasm, trapping the impermeant quin 2. In experiments with lymphocytes [29, 301 the intracellular quin 2 appeared lo be free in cytoplasm, not bound to membranes and not sequestered inside organelles such as mitochondria. In view of the pivotal role calcium plays in regulating many sperm functions 131 and its possible link with c-AMP, this paper also reports on the [Ca” +]i of ram and boar sperm after addition of dibutyry! c-AMP and the phosphodiesterase inhibitors, theophylline and caffeine which arc known to stimulate motility 131-331and to inhibit the uptake of “Ca2+ in ram and boar sperm [341. Other aspects of calcium metabolism were investigated by adding rotenone and antimycin A which prevent the stimulatory effects of calcium on phosphate accumulation by bull sperm 1351and decrease calcium and oxygen uptake in ram and boar sperm [34]. Lanthanum, an inhibitor of divalent cation uptake in rat liver mitochondria [36-381,
CEL,L CALCIUM
and carbony! cyanide-In-chlorophenylhydrazone (C’CCP) and 2,4-dinitropheno! (2,4-DNP), uncouplers of oxidative phosphorylation in mitochondria, were also tested along with sodium azide and propanolo! which inhibit the motility, but not the calcium uptake of human sperm [39]. In addition attempts were made to manipulate (Ca2+]i with the divalcnt cation ionophore A23L87, which initiates calcium influx in bull, boar, human and ram sperm (34,40,41]. Experiments were also undertaken with the polyene antibiotic blipin, a membrane disrupting agent which has been shown to release calcium accumulated in the presence of the ionophore [34,40].
Materials and Methods Collectiorl and processing of smell
Semen was collected from rams by electrical stimulation [42J and from the cauda epididymis of boars within three hours of slaughter by the method of Lasley & Bogart 1431. To obtain caput epididyma! sperm, the distal caput portion of the boar epididymis was removed and cleaned of blood vessels and connective tissue. Longitudinal incisions, approximately 5mm apart, were made and epididyma! semen extracted by gently swirling each section with medium containing 145mM NaC!, 5mM KC!, 1mM NazHP04, 1mM CaC!?, 0.5mM MgSQ, 5mM glucose and 1OmM HEPES titrated with NaOH to pH 7.4 (unless otherwise stated) at 37°C 1291. Caput semen from each section was pooled and !i!tered through 8 ply gauze (Handy) to rid the extract of tissue debris. Suspensions of the ejaculated ram and epididyma! boar spermatozoa were diluted in 5 volumes of the collection medium and centrifuged for 12 min at 500g and 2lYC. The supernatant was withdrawn, the sperm resuspended in medium and the washing procedure was repeated. The spermatozoa were !ina!!y resuspended in medium to give a concenlration of 1 x 10’ sperm/m!. Spermatozoa were counted in a haemocytometer. Motility of the sperm was estimated by light microscopy on a scale of 0 to 4 [44]. Washed spermatozoa were cold shocked by slowly pipetting
QUIN 1 AND CYTOPLASMIC
FREE CALCIUM
IN SPERMATOZOA
samples held at 25°C into glass vials maintained at 0°C in an ice bath. After 10 min all samples were brought to 30°C.
Calcium-magnesium buffers for calibration [29] were made up with Ca-Mg-EGTA at concenlrations calculated on the basis of apparent dissociation constants for Ca-EGTA and Mg-EGTA of 214nM and 8.96nM respectively at pH 7.05, 37°C (11SmM KCI, 20mM NaCl, 1OmM 4-morpholinepropanesulfonate (MOPS), 2.22mM EGTA and 1.22mM MgClz, titrated with KOH to pH 7.05 at 37°C). Fluorescence measurements were undertaken in the solution used for collection of semen. Additions of calcium were made from 1.0 or 0.2M CaCl2 stock and EGTA, pH was adjusted with 1M HCl or LM Tris base. All standard solutions were filtered through 0.45t.r.m Millipore filters prior to use and all glassware was acid washed and rinsed in a concentrated EGTA solution lo remove any contaminating calcium. Stock solutions of quin 2/AM were made up to 5OmM in dry dimethylsulphoxide (DMSO) and kept dessicated at -20°C between use. The linal concentration of DMSO from the quin 2/AM stock did not exceed 0.5% (vol/vol). A23187 was stored as a 1OmM solution in dimethylformamide/ethanol(3/1) al -20°C.
47
perature for 4 hours with no loss of quin 2 activity. For measurement of fluorescence. lml of the stock suspension was brielly centrifuged at 5OOOgand the sperm resuspended in 2ml of the fluorescent medium and transferred to the cuvette. This final step minimised the carry-over of external quin 2. The resulting quin 2 loading was approximately 3mM; a high intracellular quin 2 concentration will act as a calcium buffer and so perturb the (Ca”]i. All experiments involving quin 2 were replicated on sperm from at leas1 3 ejaculates.
Fluorescence was measured with a Perkin-Elmer LS-5 spectrofluorimeter. Standard monochromator settings were 339nm excitation with 5nm slits, and 492nm emission with 1Onm slits; 339nm was chosen as at this excitation wavelength the calcium and magnesium complexes of yuin 2 adsorb equally - and longer wavelengths would decrease the sensitivity to calcium binding. Samples were contained in lcm square quartz cuvettes thermostatically maintained at 37°C. Interruptions of the traces presented in the results show where the sample compartment was opened either to add reagents or stir suspensions with a plastic pipette. Statistical arta!vsi.s Differences between mean treatment values were assessed by t-tests.
and control
Cltertticals Sperm were loaded with quin 2 by a modificaton of the procedure described for lymphocytes by Tsein et al. 129) in the medium used for the collection and washing of sperm. Quin 2/AM (50uM) was added to the sperm suspension (10’ sperm/ml) and shaken for 20 min at 3PC in the dark. The suspension was then diluted with an equal volume of the same medium, incubated for a further 40 min (ram sperm) or 60 min (boar sperm), centrifuged at 1OOOgfor 4 min and the sperm resuspended in fresh medium. This suspension could be kept in the dark at room tem-
Quin 2 and quin 2/AM were obtained from Calbiochem, La Jolla, California, USA; A23187 and anlimycin A from Boehringer Mannheim, Mannhcim, West Germany; caffeine, propranolol, sodium azide, rotenone, dibutyryl c-AMP, theophylline and carbonyl cyanide-ln-chlorophenylhydrazone (CCCP) from Sigma Chemical Co., St Louis, Missouri, USA; 2,4_dinitrophenol (2,4DNP) from Ajax Chemicals, Sydney, Australla. Filipin was a gift from Upjohn International Inc., Kalamazoo, Michigan, USA. All other chemicals were of analytical reagent grade.
48
CELL
CALCIUM
Results Spectra of qttin 2 ii; presence of calcium
Preliminary experiments were undertaken to check that the quin 2 method of Tsein et al. [29] was suitable for measuring intracellular calcium in sperm. The emission spectra of quin 2 were observed in the presence of free Ca2+ ranging from a concentration of ‘0’ (< 1nM) to a saturation level of 10Ot~M. The solutions were Ca*+-EGTA buffers with concentrations of free cations chosen to mimic the intracellular environment. With excitation at 339nm, the intensity of fluorescence increased about six-Cold over a range of [Ca’+] from 0 to lOOp,M although the wavelength of the emission peak remained nearly constant at 492nm. The fluorescence response was nearly maximal for calcium concentrations above lt~,M. The dependence of fluorescence at 492nm on free-calcium was checked for this system and the results conformed to the theoretical behaviour of a simple 1 dye: 1 Ca” + binding with an effective dissociation constant of 115nM. The absence of free Mg*+ resulted in an effective dissociation constant of 60nM as found by Tsein et al. [29].
%
s
2 P
I’.
;
~~~_
J1-Pom’n
i
_/
400
I
450
500
1 550
I 800
mm
E’ig. 2
Shifts of emission spectra during quin 2/AM
uptake
and hydrolysis in ram sperm. Excitation 339n?;The initial fluorescen e of quin 2/AM is shown in ImM Ca solution and s then 10 speml were added. Dashed curves show the suspension spectra at 20 and 60 min. The curve peaking at 492nm was obtained after washing the sperm. resuspending in the same volume of fresh medium then releasing the trapped dye with Triton X-100. The bottom trace ‘sperm alone’ shows the autofluorescence measured from unloaded cells at the same density
The emission spectra
of 20uM quin 2 at
‘O’Ca* + ( c lnM), 2OOnM Ca*+ and 1mM Ca*+
are shown in Figure 1 at pH 6.75, 7.05, 7.50 and 8.00. The [Ca*+] was altered by appropriate additions of Ca-EGTA, CaClz and K-MOPS while carefully adjusting the pH. The spectra at ‘0’ Ca” ’ were unaffected by pH change but at 1OOnMCa*+, lowering the pH from 7.05 to 6.75 inhibited fluorescence. However, raising the pH to 7.5 and 8.0 had little effect on the emission spectra. It is important to note that the effect of pH on the affinity of quin 2 for calcium is difficult to study because EGTA is much more pH-dependent than quin 2, thus in latter experiments in which pH was altered very careful adjustments were necessary. Loading of quirt 2 into spem 400
410
500
IS0
000
of quin 2/AM in sperm was easily monitored by the gradual shift in the emission spectrum from that of the ester which peaks at 430nm, to that of quin 2 itself peaking at 492nm. This is shown in Figure 2 for an experiment on ram sperm in which
Hydrolysis
Fig. 1 (cInM
Ey$sion spectra $ri: 20f.tM quin 2 at ‘0’ Ca2+ Ca ), IOOnM Cy: and 1mM Ca*+ at pH 6.75. ] and pIi were altered by appro-EGTA.
careful adjustment of pH
CaC12 and K+-MOPS
with
QUIN 2 AND CYTOPLASMIC
FREE CALCIUM
49
IN SPERMATOZOA
Table 1 Motility scores (+ S.E.M.) for three observations of ejaculated ram and cauda boar sperm in the presence and absence of 3mM quin 2 Sperm
Addition
0
lhr
Ejaculated ram
Control Quin 2
3.6 2 0.3 2.6 2 0.3
3.4 -+ 0.3 3.2 f 0.2
3.4 + 0.3 3.2 + 0.1
3.0 2 0.1 2.9 ? 0.2
Cauda boar
Control Uuin 2
3.2 + 0.1 3.2 ? 0.1
3.2 + 0.2 3.2 f 0.1
3.0 -t 0.2 2.9 +- 0.1
3.0 f 0.2 2.8 ‘-r-0.1
5OpM quin 2lAM was added to 10’ sperm/ml; the dashed curves show the emission spectra at 20 and 50 min by which time the spectral shift was almost complete. A large proportion of the quin 2 was trapped inside the sperm, as demonstrated by centrifuging and resuspending the sperm in fresh medium to remove external quin 2. It took longer (approx. 80 min) to load boar epididymal sperm with quin 2 but the curves were similar to ram sperm (Fig. 2). Because the dye measures [Ca”+] by a change in fluorescence intensity, the signal depends on [Cal+] as well as the dye content of the suspension and in order to measure the [Ca’ + 1in sperm it was necessary to mix the dye with known levels of Ca’+ while keeping the dye content of the suspension constant. This was done at the end of each experiment by releasing the dye from sperm with Triton X-100. The medium contained 1mM Ca” so that maximum fluorescence (Fmax) was obtained after releasing the dye. [Ca”+] was then set at 300 and 1OOnM by adding EGTA and MgCl2. Minimum fluorescence (Fmin) was obtained by adjusting the calcium concentration to 1nM by addition of 2nM EGTA and enough Tris base to take the pH to >8.3. The autofluorescence of unloaded sperm was also taken into account and an appropriate correction made to Fmaxand Fmin. The sperm regenerated quin 2 with the correct Ca’+ affinity and it was sufficient in routine experiments to note the Fmax and Fmin values in the lysate and calculate the [Ca’+]i from intact cells by substitution in the following equation [29]: (Ca”+l = Kd (F- F,in)/(F,,,-
F)
21Ir
3111
Fluorescence observations in several ex eriments indicated a normal intracellular [Ca 2-F) ] of 193nM +- 0.2 (n = 10) for ejaculated ram sperm, 175nM -c 3.9 (n = 10) for cauda boar sperm and more variable levels of 1lOnM 5 10 (n = 10) for caput boar sperm. Table 1 shows that quin 2 was not toxic to ejaculated ram and cauda epididymal boar sperm over 3 h as gauged by motility. As the movement of caput boar sperm was restricted to slight twitching before and after addition of quin 2, no results are presented. It is evident from Table 1 that the motility of ram and boar sperm is well sustained in the saline medium employed. If ram ejaculated or boar cauda sperm were first cold shocked at 0°C they could not be loaded subsequently with quin 2/AM. Furthermore, if normal sperm were loaded with quin 2/AM, exposure to cold shock caused slow release of quin 2 into the medium. In contrast, cold shocked caput boar sperm could be easily loaded with quin 2 and had a [Ca”+]i of 117 + 8.8nM (mean + S.E.M.; n =4) which was similar to the 121 ? 6.9nM of control sperm. Effect of inhibitors, activators and swfactam
Figures 3 and 4 show the effect of metabolic inhibitors, activators and surfactants on the intracellular calcium levels of ejaculated ram, and cauda and caput boar sperm. The inhibitor sodium azide and the B-adrenergic antagonist, propranolol, which abolished the motility of sperm from both species, had no significant effect on the [Ca”]i of the sperm. Similarly the phosphodiesterase inhibitor caf-
50
CELL CALCIUM nY
1OmY
6mY
Na
Ca
2+
AZIDE _
196
_ -
206 192
CAFFLIWE
SmM
nM
DIBUTYRYL
Cm’+
1.2mMPROPI1ANOLOL r
,102
6mM I
THtDPHYLLlNC
CAMP
-1,)
2#Y CCCP
-220 -1e3
\ luM AWTIYYCIN
I
6 WIN
2.6mY
A
-106
L.)+
I
Fig. 3 Reproductions of spectrofluorimeter of ejaculated ram sperm
traces showing the effect of substances on the cytoplasmic free calcium concentration
feine which stimulates motility in boar caput sperm (341 produced no significant change in calcium levels of the sperm of either species. By contrast the other phosphodiesterase inhibitor, theophylline, and dibutyryl c-AMP produced a ~~dual but significant (P ~0.01) decrease in [Ca ]I in both ram and boar sperm. The decreases in [Ca*+]i (mean -c S.E.M., n=3 or 4) after 5 minutes exposure were: ram, 193 + 0.3 to 107 f 5.6 (theophylline), 193 + 0.4 to 91 f 0.9 (dibutyryl c-AMP); boar cauda, 175 f 4.1 to 98 f 8.1 (theo-
phylline), 174 f 3.8 to 119 f 8.1 (dibutyryl cAMP); boar caput, 113 + 1.2 to 54 + 3.1 (thcophylline), 118 f 2.1 to 56 f 7.8 (dibutyryl cAMP). The inhibitors rotenone and antimycin A, and the uncouplers of oxidative phosphorylation, CCCP and 2,4-DNP, produced a transient elevation of [Ca*+ Ii with a subsequent return to control levels or below. Addition of La3+ caused a rapid decrease (P < 0.05) in the [Ca*+]i of ram and boar sperm to a
WIN
2 AND CYTOPLASMIC
FREE CALCIUM
CAUDA
BOIR
IN SI’ERMATOZOA BOAR
CAPUT
SPERM
SPERM
ClY c.‘+ lomY
F
Ya
1
C.2+
AtlOf!
-1T6
l.¶mY
w 1
lnoPaANoLoL
l.tmM
-116
6mY
p
8mY
1
-110
CAFFEINE
A
T~LO?liVLLlN6
-110 IMY
-I?6
DIBUTVRYL
PROCRAWOLCL
6mY
CACCSINI
-1t6
5mY I
nN I(8
AZIDE
CAMP
--(‘O
THLOPHY
LLINL
,--!-+-L_ 1’:: SKY
DIBUTYRYL
CAMP
1’::
-i__ SUN
,6uN
ROTLWOWC
ANTIMVCIN
L
‘It6 SOYY
A
-
60
-
26
1.4-OWC -166 -176
L
\ 2.6mN
-
60
I..*+
2.6mN
1
L.‘+
-176 --t,____
Fig. 4 Reproductions of spectrofluorimeter tion of ejaculated boar spermatozoa
tracings
showing the effect of substances
Y7:
on the cytoplasmic
free-calcium
concentra-
52
CELL
EJACULATED
RAM SPERM
CAUDA nY
A.pH
Ca
BOAR
SPERM
II
nM Ca
CAPUT
21
BOAR
CALCIUM
SPERM nM Ca
r --
7.5
I+
450
i
I’ t
A23187
A23187
-3
-183
/ A23187
d -173
B.pH
Ii
\
8.0
-__i
-123
--7
-450
r+_._.w-
/
(
i
A23187
I
A23187
d1
-188
v
A231871
+..“.bJ
-180
kid -I
Fig.
5 Reproductions
calcium concentration
of spectrofluorimeter
-563
/
-440
-127
traces showing the effect of the ionophore A23187 (3.0l~,M) on the cytoplasmic free-
of ram and boar sperm incubated at (A) pH 7.5 and (B) pH 8.0
, 0
4
8
CONCENTRATION
12
16 OF
20 A23187
24
28
0
(#MI
Fig. 6 The effect of increasing concentrations of the ionophore A23187 on the cytoplasmic free-calcium concentration of ram sperm incubated at pH 8.0. Each point represents the mean f S.E:M. for 3 experiments
. 4
8
CONCENTRATION
Fig. 7
12
10 OF
20 A23187
The effect of increasing concentrations
24
28
(MM)
of the iono-
phore A23187 on the cytoplasmic free-calcium of cauda (triangles) and caput (squares) boar sperm incubated at pll 8.0. Bach point represents the mean k S.E.M. for 3 experiments
QUIN
2 AND
CYTOPLASMIC
FREE
CALCIUM
low level that was maintained throughout the exThe decreases in (Ca-+li after 5 periment. minutes exposure were: ram, 193 + 0.4 to 161 r 5.9; boar cauda, 175 f 3.7 to 102 + 6.2; boar caput, 114 + 3.1 to 71 + 16.7. Effect of the ionophore A23187
It was noted in an earlier study 1451that the ionophore-induced accumulation of “Ca”+ by ejaculated and cauda ram sperm increased under alkaline conditions but in boar sperm the calcium increase was less sensitive to changes in pH. Quin 2 measurements (y$. 5) indicated a large (P < 0.01) increase in [Ca ]i from 1%3 + 6.5 to 450 + 18.9nM (n = 4) in ram sperm at pH 8.0 on addition of 3.0t~,MA23187 compared to a smaller rise at pH 7.5. Once again raising the pH of the medium had little effect on the large increase in [Ca’+]i of boar cauda and caput sperm incubated with the ionophore. The effect of increasing concentrations of A23@7 on the [Ca”]i of ram and boar sperm at pH 8.0 are shown in Figures 6 and 7 respectively. The lower ionophore concentrations (0.33 to 3.0t~_M)rapidly increased (Ca”]i (P
RAM SPERM
53
IN SPERMATOZOA A.
EJACULATED
RAM
0.
CAUDA
SPERM
3YY
BOAR
C.
CAPUT
Fig. 8
BOAR
Reproductions
-200 *1
SPERM
of spectrofluorimeter
CAUDA BOAR SPERM
CAPUT BOAR SPERM
nM
ha’
nM Cm” FILIPIN
-176 +FILIClN
-_-
--._--
-273
--_I-
-420
-102 FILIPIN
r-i
A29
j
I’;:
k-
Fig. 9 (3.0&M)
traces showing
nophore to ram and boar sperm was apparently not dependent on oxidative metabolism of the mitochondria, as subsequent addition of the inhibitors CCCP and 2,4-DNP did not affect [Ca’+ Ii. In an attempt to detect any calcium stored in
-307
-193
-120
the effect on the cytoplasmic free-calcium concentration of adding (he ionophore A23187 to ram and boar sperm in calcium free medium
FILlPIN
-380
nM c.*+
A23187
L/-l_
nM CI~’
FILlPIN
SPBRM
Reproductions of spectrofluorimeter traces showing the effects of (A) filipin (0.2mM) plus filipin. on the cytoplasmic free-calcium concentration of ram and boar sperm
-
15
and (U) the ionophore
A23187
54
the mitochondria, or indeed any membrane-bound organelle, 3.0uM A23187 was added to ejaculated ram and epididymal boar sperm in CaZ+-free medium (Fig. 8) and resulted in a transient rise in [Ca’+ Ii. The polyene antibiotic filipin, forms a complex with cholesterol in biological membranes [46, 47) while leaving the mitochondria intact [40, 481. It increased (P < 0.01) [Ca’+Ji in ejaculated ram, and cauda and caput boar sperm (Fig. 9). However, 0.2mM filipin released calcium from ram and boar sperm previously exposed to ionophore which had increased [Ca’+]i. In the presence of the ionophore, the cytoplasmic free-calcium concentration was not affected by theophylline, rotenone or antimycin A, all of which altered the [Ca’+[i of control sperm. However, addition of La3+ to ionophore treated ejaculated ram and cauda boar sperm decreased (P ~0.01) [Ca”+ Ji from 420 f 9.lnM to 210 f 6.4nM and from 530 f 8.lnM to 170 + 13.lnM. These values did not differ significantly from those recorded for control sperm.
Discussion Quin 2 can be easily loaded into ram and boar spermatozoa as the acetoxymethyl ester which then hydrolyses. Loadings of several mM are not toxic to these spermatozoa as judged by motility scores and it has proved a suitable indicator of [Ca”+ Ji. The values obtained are similar to those for human sperm [21]. It has been shown that cold shocking ejaculated ram [19] or cauda boar sperm [34] causes damage to the plasma membranes and to the acrosome, and this structural damage would account for the failure of shocked ejaculated ram and cauda boar sperm, to retain quin 2. In contrast, caput boar sperm do not suffer the same structural damage on cold shocking to 0°C (341 and can therefore be readily loaded with quin 2 and have an internal calcium concentration similar to conlrol sperm. The concentration of inhibitors and pharmacologically active substances tested in these experiments are within the range required lo elicit their physiological effects [49]. However, it should be
CELL CALCIUM
remembered that in media containing glucose, spcrmalozoa can probably maintain their ATP by glycolysis and respiratory inhibitors may therefore, under these conditions, have little effect on the ATP concentration in the sperm or the Ca” ’ /Mg2 + ATPase of the plasma membrane. The metabolic inhibitor, sodium azide, and the surface active a ent, propranolol severely inhibited the uptake of 49Ca”+ by ram and boar sperm 134). However, the quin 2 fluorescence measurements suggest there is no change in [Ca2+]i. Although caffeine had no effect on the [Ca2+li of ram and boar sperm, theophylline and dibutyryl c-AMP gradually reduced calcium levels in a comparable fashion lo the inhibition of 45Ca2+ uptake (491. These results support the proposal of Peterson et al. [41] that theophylline and dibutyryl cAMP may act on the plasma membrane calcium pump through an interaction with c-AMP to reduce internal calcium levels in the sperm. As large increases in internal calcium are detrimental to the sperm, this may be a mechanism by which cyclic nucleotides maintain active motility. The transient elevation of sperm [Ca2+]i on adding the inhibitors, rotenone and antimycin A, and the uncouplers, CCCP and 2,4-DNP, was most likely the result of release of mitochondrial calcium stores. The return lo control levels or below was probably due to the extrusion of released calcium via the plasma membrane calcium pump. The addition of La3+ resulted in slow leaching out of internal calcium, presumably by disruption of normal calcium transport. The results obtained on adding low concentrations of the calcium ionophore A23187 to sperm were as expected 134, 40, 411 and its action of increasing [Ca’+]i was highly pH dependent in ram sperm in contrast to boar sperm. Quin 2 could not be used to monitor the rise in [Ca2+]i in the presence of high concentrations of the ionophore due to saturation of the dye. Apparently, the large increase of [Ca’+]i in response to A23187 was not dependent on the oxidative metabolism of the mitoc.hondria as subsequent addition of the uncouplers, CCCP and 2,4-DNP, had no effect on sperm [Ca’+]i in either species. The increase in the [Ca2+]i of ram and boar sperm induced by filipin is in agreement with other studies based on
QUIN
2 AND
CYTGPLASMIC
FREE
CALCIUM
55
IN SPERMATOZOA
murexide and 45Ca2+ uptake [34,40,41]. Addition of lilipin to ionophore treated ram and boar sperm, however, decreased ]Ca2+Ji. This would not be expected if there was gross disruption of the plasma membrane by filipin and similar results with bull sperm have been interpreted as evidence for lack of mitochondrial involvment in the calcium influx induced by A23187 1401. Addition of La3+ also reduced the ionophore-induced increase in [Ca’+]i, possibly by reaction with calcium carrier sites and disruption of the balance struck between the intcracting mitochondrial and plasma membrane calcium pumps. The increase in [Ca”+]i produced when A23187 was added to sperm in calcium-free medium quickly returned to control levels, most likely through extrusion by the plasma membrane calcium pump. The quin 2 measurements of cytoplasmic-free calcium in ram and boar sperm in the presence of A23187 support the suggestion of Babcock et al. [4O] that high concentrations of calcium and ionophore overwhelm both calcium membrane pumps and calcium enters a non-mitochondrial compartment of the sperm as a consequence of the equilibration of the ion across both mitochondrial and plasma membranes. The sensitivity of (Ca’+ ]i to filipin indicates that calcium binding to soluble intracellular components may also be involved 140, 411. The advantages of quin 2 as an indicator of [Ca2’]i in ram and boar sperm are that it can be non-disruptively loaded into the cells, it is nontoxic to the spermatozoa and it seems IO be as good or better than other reagents for quantifying calcium levels below lo-‘M. There is, however, a problem with autofluorescence in some sperm preparations and it is difficult to quantitate calcium levels above lt.~M as seen after addition of the ionophore. Similar problems have been encountered in measuring the [Ca2+]i of lymphocytes ]2Y]. Acknowledgements
The authors are indebted to Dr. 1. Kennedy for advice in setting up the assay, Dr. W. Jones for use of a spectrofluorimeter, Miss K. Murdoch for her invaluable technical assistance and to the Australian Wool Corporation for linancial support (I.G.W.) and a studentship (A.M.S.).
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Ca
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Please send reprint requests to: Dr. LG. White, Department of Veterinary Physiology. University of Sydney. 2006, N.S.W., Australia. Received: 19 May 1987 Accepted: 6 January 1988