Effect of cold shock and cooling rate on calcium uptake of ram spermatozoa

Animal Reproduction Science, 12 (1986) 131--143

131

Elsevier Science Publishers B.V,, Amsterdam -- Printed in The Netherlands

EFFECT, OF COLD SHOCK AND COOLING RATE ON CALCIUM UPTAKE OF RAM SPERMATOZOA

ANN M. SIMPSON and I.G. WHITE

Department of Veterinary Physiology, University of Sydney, Sydney 2006, N.S. W. (Australia) (Accepted 28 May 1986)

ABSTRACT Simpson, A.M. and White, I.G., 1986. Effect of cold shock and cooling rate on calcium uptake of ram spermatozoa. Anita. Reprod. Sci., 12: 131--143. Rapid cooling (cold shock) of washed ejaculated ram sperm irreversibly reduced motility and respiration and greatly increased uptake of 4sCa2+. The effect was greater as the temperature of cooling was reduced from 15°C to 0°C, and a substantial increase in sperm calcium levels was even observed after slow cooling to temperatures below 10°C. The rise in calcium uptake on freezing sperm to -79°C was not as great as that on cold shocking sperm to 0°C. Inactivation of sperm by mild heat (50°C) had no significant effect on calcium uptake but subsequent cold shock increased the sperm calcium. Reverse immobilization of sperm by low concentrations of formaldehyde significantly reduced calcium uptake on cold shock. Addition of detergents to sperm immediately reduced motility, respiration and calcium uptake of control and cold-shocked sperm to zero.

INTRODUCTION Calcium is i m p o r t a n t in t h e r e g u l a t i o n o f cell f u n c t i o n as it acts as a " m e s s e n g e r " t h a t c o - o r d i n a t e s a h o s t o f intracellular r e a c t i o n s ( L e h n i n g e r et al., 1 9 6 9 ; Berridge, 1 9 7 5 ; Garbers a n d K o p f , 1 9 8 0 ) . A n o p t i m a l c a l c i u m c o n c e n t r a t i o n is a p p a r e n t l y m a i n t a i n e d in m a t u r e m a m m a l i a n s p e r m b y the i n t e r a c t i o n o f m e m b r a n e p u m p s associated w i t h t h e p l a s m a m e m b r a n e and t h e m i t o c h o n d r i a . I t has b e e n suggested t h a t t h e m i t o c h o n d r i a l p u m p a c c u m u l a t e s c a l c i u m ( B r a d l e y et al., 1 9 7 9 ) . w h e r e a s t h e p l a s m a m e m b r a n e s y s t e m p u m p s c a l c i u m o u t w a r d s via a Ca2+-dependent Mg2+-ATPase ( B r a d l e y a n d F o r r e s t e r , 1 9 8 0 ; B r e i t b a r t a n d R u b i n s t e i n , 1 9 8 3 ; B r e i t b a r t et al., 1 9 8 3 , 1984). T h e u p t a k e o f c a l c i u m b y m a m m a l i a n s p e r m ( B a b c o c k et al., 1 9 7 6 ; P e t e r s o n et al., 1 9 7 9 ) has been s h o w n t o be involved in t h e a c r o s o m e react i o n (Green, 1 9 7 8 ; P e t e r s o n et al., 1 9 7 8 ; J a m i l a n d White, 1 9 8 1 ; ShamsB o r h a n a n d Harrison, 1 9 8 1 ) , a c t i v a t i o n o f a d e n y l a t e cyClase a n d i n d u c t i o n o f m o t i l i t y ( M o r t o n et al., 1 9 7 3 ; H y n e a n d Garbers, 1 9 7 9 a , b; K o p f and

0378-4320/86]$03.50

© 1986 Elsevier Science Publishers B.V.

132 Garbers, 1980). On the other hand, too high a concentration of calcium in the external medium decreases the motility of sperm of many species, presumably by excessively raising intracellular calcium levels (Rosada et al., 1970; Quinn et al., 1970; Brokaw et al., 1974; McGrady et al., 1974; Davis, 1978). When the sperm of the ram, bull and several other species are cold shocked by rapidly cooling to 0°C, motility and metabolic activity are irreversibly decreased, the plasma membrane disrupted and the permeability altered (Blackshaw, 1954a; Mann and Lutwak-Mann, 1955; Blackshaw and Salisbury, 1957; Wales and White, 1959; Qulnn and White, 1966; Quinn et al., 1969; Karagiannidis, 1976; Glover and Watson, 1985). Atomic absorption spectrometric analyses suggest that these cold shock changes may be accompanied by an increase in the calcium content of the sperm (Quinn and White, 1966; Karagiannidis, 1976). In the present study, the possible link between calcium intoxication and cold shock has been further explored by measuring the progressive uptake of radio-active calcium into ram sperm cooled slowly, rapidly and snapfrozen. Comparative studies have also been made of ram sperm exposed to mild heat and detergents which would be expected also to disrupt the membranes. Ram sperm have also been treated with low concentrations of formaldehyde (Dott and Foster, 1975) which has been shown to reduce the susceptibility of bull sperm to cold shock, presumably by stabilizing the plasma membranes (Karagiannidis, 1976), and their calcium uptake measured. MATERIALS AND METHODS

Sperm Semen was collected from rams by electrical stimulation using the technique of Blackshaw (1954b). Samples with high sperm motility were diluted in five volumes of the incubation medium (250 mM sucrose, 5.0 mM HEPESTris, 5.0 mM succinic acid-Tris, 0.3 mM sodium dihydrogen phosphate) pH 7.4 (Van Eerten et al., 1978) and centrifuged for 12 min at 500 g and 20°C. The washing procedure was repeated to remove residual seminal plasma and the spermatozoa were resuspended in the incubatioa medium to give a concentration of 1 X 10S/ml. Spermatozoa were counted in ahaemocytometer. Washed spermatozoa were cold schocked by slowly pipetting samples held at 25°C into glass vials maintained at 0°C in an ice bath or 5--15°C in a water bath. Sperm was frozen in a dry-ice alcohol mixture. After 10 rain all samples were brought to 30°C. For slow cooling, test tubes containing the sperm suspensions were immersed in a large beaker of water at 25°C and cooled to 0, 5, 10 or 15°C over a period of 3 h (drop of 2°C every 15 min) by adding ice and stirring. Samples were then returned to 30°C. Three detergents, Triton X-100, sodium dodecyl sulphate (SDS) and

133

N-cetyl-N, N, N-trimethylammonium bromide (CTAB) were dissolved in the incubation medium and added, at a final concentration of 0.01 and 0.1%, to control and cold-shocked suspensions of ram sperm at the beginning and after 24 min of incubation with 4SCa2÷. Two other methods were employed to render the sperm immotile: (1) heating sperm suspensions at 50°C for 10 min, (2) adding formaldehyde to give a final concentration of 0.025%. The latter abolishes motility which can be restored by removing the formaldehyde by dialysis (Dott and Foster, 1975). Heat- and formaldehyde-treated sperm were then cold shocked as previously described. The motility of sperm was scored under a light microscope on a scale of 0 to 4 (Emmens, 1947).

Calcium uptake 4SCa2÷ uptake of sperm was determined using a radiochemical procedure modified from Babcock et al. (1975), Van Eerten et al. (1978) and Peterson et al. (1979). Washed spermatozoal suspensions were equilibrated at 30°C for 5 min. 4SCa2÷-HEPES-Tris was added to give a final concentration of 1 mM, 2 pCi/ml, and the sperm incubated at 30°C. Aliquots of 0.1 ml were taken at intervals and rapidly mixed in separate test tubes with 3 ml of buffer containing 250 mM sucrose, 5.0 mM HEPES-Tris, 0.3 mM sodium dihydrogen phosphate and 10 mM CaC12 (pH 7.4) at 30°C and filtered through glass fibre disks (Whatman GF/C) at low vacuum. The filters were rapidly washed five times with buffer, dried and the radioactivity determined in a toluene-Brydet scintillation fluid.

Oxygen uptake Respiratory activity of the washed sperm suspensions was determined at 30°C using an oxygen electrode (Rank Bros., Bottisham, Cambridge) connected to a chart recorder (Leeds Northrup, Speedomax). The oxygen uptake (pl/h per 108 sperm) was calculated from the slope of the linear trace.

Chemicals 4SCa2+ w a s supplied by Amersham International, U.K., CTAB by Merck AG, Darmstadt, Germany, SDS by Sigma Chemical Co., St. Louis, MO, U.S.A., Triton X-100 by H.B, Selby & Co., Sydney, Australia, and HEPES (N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid) by CalbiochemBehring Corp., La Jolla, CA, U.S.A. All other chemicals were of analytical reagent quality.

Statistical analysis Motility scores and oxygen uptake values were analysed by t-tests, and

134

calcium u p t a k e data by a r a n d o m i z e d c o m p l e t e block split plot analysis of variance after a ln(x + 1) t r a n s f o r m a t i o n due to h e t e r o g e n e i t y of variance. RESULTS

Effect of cold shock and freezing Th e u p tak e of 4SCa2+ by washed ram sperm (Fig. 1) was linear over the first 12 rain, it increased t o a plateau of 28.2 + 7.4 nmoles Ca~÷/10 s sperm at 24 min and this level was maintained over the 200 rain of the experiment. By contrast th e sperm which had been cold shocked t o 0°C showed a significantly higher (P < 0.01) rise in calcium u p t a k e at 24 min t o a level of 147.0 + 1.0 nmoles Ca2÷/108 sperm, which subsequently fell rapidly over the course of the experiment. When ram sperm were deep frozen (Fig. 1), a calcium u p tak e curve similar in appearance to cold shock was generated; however, th e peak was significantly (P < 0.05) lower at 79.1 + 14.5 nmoles Ca2÷/10 s sperm t han cold-shocked sperm and rapidly fell t o 24.1 + 6.0 nmoles Ca2÷/108 sperm, a value n o t significantly different f r o m t h a t rec o r d ed f o r c o n t r o l sperm. Subsequent cold shocking of sperm which had previously been f r ozen did n o t alter calcium levels f r o m those of sperm only subjected to freezing.

fE

11

w 12 o. O

O

ED

8

Ih

O

•4 0

0 40

~ 80

MN IUTES 120

160

200

Fig. 1. Effect of cold shock to 0°C and snap-freezing to -79°C on the uptake of 45Ca~+ by washed ram spermatozoa. Vertical bars (sometimes omitted for clarity) represent the S.E.M. for 3 observations. Th e mo tility of sperm r ew a r m e d following cold shock t o 0°C was very low; 0.5 + 0.1 (n=3), c o m p a r e d t o a value o f 3.6 -+ 0.6 for c o n t r o l sperm. Similarly th e o x y g e n u p t a k e of cold-shocked sperm, 8.2 + 0.2 ~l/h per 108

135

sperm (n=3), was significantly (P < 0.001) lower than the value obtained for control sperm, 24.0 + 0.1 #l/h per 10 ~ sperm. The motility and respiratory capacity of frozen sperm was zero.

Rate of cooling The effect of the rate of cooling on the response of ram spermatozoa to cold shock was investigated in more detail. Rapid cooling of sperm to 0, 5, 10 and 15°C (Fig. 2) significantly increased (P < 0.01) calcium uptake compared to control sperm. As might be expected, calcium uptake increased with increasing temperature gradients, the highest value of 190.1 -+ 10.5 nmoles Ca2÷/10 s sperm at 24 min inevitably being recorded for sperm rapidly cooled to 0°C. However, the calcium uptake of sperm cold shocked at 5°C was not significantly higher than sperm shocked at 10°C. Surprisingly, when the same sperm were very slowly cooled to between 15 ° and 0°C, 45Ca2÷ uptake (Fig. 3) values were significantly higher ( P < 0.01) than the control in all cases except sperm slowly cooled to 15°C. A comparison of Figs. 2 and 3 shows that the 45Ca2+ uptake curves of sperm slowly cooled to ! 0 , 5 or 0°C are very similar to t h e curves of identical sperm rapidly cooled to these temperatures. Table 1 shows the motility score and oxygen uptake of washed ram sperm 200,

160 o~ uJ m¢'~ 1 2 0 0

m ~ w -I 0

80

I 40

2'0

4'0 6'0 MINUTES

8'0

100

Fig. 2. Effect of rapidly cooling to 15, 10, 5 and 0°C on the uptake of *sCa2÷ by washed ram spermatozoa. Vertical bars (sometimes omitted for clarity) represent the S.E.M. for 5 observations.

'60t

136

JO

~+~ 8 0 C) LU .J 0 :E : 4

0

0

20

~

40

60

80

MINUTES

100

Fig. 3. Effect of slowly cooling to 15, 10, 5 and 0°C on the uptake of 4SCa~* washed ram spermatozoa. Vertical bars (sometimes omitted for clarity) represent the S.E.M. for 5 observations. TABLE 1 Motility and oxygen uptake (mean + S.E.M. for 3 observations) of washed ram spermatozoa returned to room temperature after cooling rapidly or slowly Cooling temperature (°C)

Rapid cooling Motility score

O~ uptake (/~l/h per l 0 B sperm)

Motility score

02 uptake (/~l/h per l 0 B sperm)

Control (25) 15 10 5 0

3.8 *'1.5 ~**1.0 ***0.7 ***0.3

25.5 *'13.3 *'11.2 **7.6 ***5.9

3.8 *3.2 "2.8 *'1.9 *'1.5

25.5 24.9 *'21.1 *'13.2 **8.8

+ 0.08 -+ 0.14 + 0.08 + 0.08 + 0.14

Slow cooling

+ 0.65 + 0.57 + 0.64 -+ 0.36 + 0.52

± 0.08 +- 0.08 ± 0.08 • 0.05 +- 0.14

+ 0.65 -+ 0.95 + 0.73 +- 0.66 + 0.44

*P < 0.02; **P < 0.01; ***P < 0.001.

r e t u r n e d to r o o m t e m p e r a t u r e after cooling r a p i d l y or slowly to 15, 10, 5 a n d 0°C. T h e r e w a s a p r o g r e s s i v e d e c r e a s e i n b o t h p a r a m e t e r s w i t h i n c r e a s i n g temperature gradient and although the effects were greater after rapid cooling, motility and oxygen uptake were significantly depressed on slow c o o l i n g b e t w e e n 1 0 a n d 0°C.

137

Effect of heating to 50°C Heating suspensions of washed ram spermatozoa at 50°C for 10 min caused irreversible loss of mobility and respiratory activity. However, heating did n o t greatly increase calcium uptake of the sperm (Fig. 4). U p t a k e was much less than after cold shock, and when heated sperm was subsequently cold shocked the further increase in calcium uptake was n o t statistically significant. 180.

• 140. m

~

H

E

A

T

E

D

2o +~ 1 0 0 . m

60

20 0

40

80 120 MINUTES

160

200

Fig. 4. Effect of cold shock to 0°C, heating to 50°C and cold shock to 0°C following heat treatment, on the uptake of 4SCa2+ by washed ram spermatozoa. Vertical bars (sometimes omitted for clarity) represent the S.E.M. for 3 observations.

Formaldehyde treatment When ram spermatozoa were temporarily immobilized b y a low concentration of formaldehyde, the calcium uptake of the spermatozoa held at r o o m temperature or subsequently cold shocked to 0°C was significantly lower (P < 0.01) than that of control sperm (Table 2). If the f o r m a l d e h y d e was removed b y dialysis, the calcium uptake of both control and cold-shocked sperm rose to the levels of the control sperm that had n o t been exposed to formaldehyde. The calcium uptake of sperm cold shocked w i t h o u t formaldehyde was significantly higher (P < 0.01) than all other values. The motility and respiratory activity of control and cold-shocked sperm was zero in the presence of formaldehyde b u t returned to control levels o n

138 TABLE2 M o t i l i t y , o x y g e n u p t a k e , a n d u p t a k e o f *SCa2+ a f t e r 2 4 m i n ( m e a n + S.E.M. f o r 3 observations) of washed ram spermatozoa returned to room temperature after cold shock and exposure to formaldehyde Treatment

Motility score

Control Cold s h o c k Formaldehyde Formaldehyde cold s h o c k aFormaldehyde removed aFormaldehyde removed, then cold s h o c k e d

Oxygen uptake ( ~ l / h p e r 108 s p e r m )

Calcium uptake ( n m o l e s Ca2÷]108 s p e r m )

+ 0 + 0.08 +- 0 -+ 0

31.9 **'6.1 ***0 ***0

+ 0.16 + 0.15 -+ 0 -+ 0

52.9 *'124.8 *'16.7 *'19.5

3.8 + 0.05

29.9

+ 0.12

4.0 0.4 ***0 ***0

3.6 + 0.08

* ' 1 1 . 2 5 + 1.0

+ 8.90 + 16.98 + 3.00 + 2.57

55.6 +

6.2

58.3 + 1 0 . 4 2

a S p e r m were i n c u b a t e d for 10 m i n w i t h 0 . 0 2 5 % f o r m a l d e h y d e a n d t h e ' f o r m a l d e h y d e r e m o v e d b y dialysis ( D o t t a n d F o s t e r , 1 9 7 5 ) . **P < 0.01; ***P < 0.001.

'°°

+~ % O

4

J

0 2

w

0

2

4 0'

6 ~0

'

80

MINUTES

Fig. 5. T h e e f f e c t o f a d d i n g s o d i u m d o d e c y l s u l p h a t e (final c o n c e n t r a t i o n 0.01%) o n t h e a c c u m u l a t i o n o f 45Ca2÷ b y w a s h e d r a m s p e r m a t o z o a . A f t e r 24 rain i n c u b a t i o n s o d i u m d o d e c y l s u l p h a t e was a d d e d t o h a l f t h e s p e r m s u s p e n s i o n a n d t h e o t h e r h a l f was r e t a i n e d as a c o n t r o l . V e r t i c a l b a r s ( s o m e t i m e s o m i t t e d f o r c l a r i t y ) r e p r e s e n t t h e S.E.M. f o r 3 observations. (o) C o n t r o l ; (A) Cold s h o c k e d .

139 removal b y dialysis. Although sperm cold shocked after removing formaldeh y d e had an appreciable oxygen uptake, it was not as great as that of the unshocked sperm.

Action of detergents If after incubating with 4SCa2+ for 24 min SDS (final concentration 0.01%) was added to control or cold shocked (0°C) ram spermatozoa, there was an immediate efflux of the accumulated calcium (Fig. 5). Addition of CTAB and Triton X-100 had the same effect. When the detergents were added prior to 45Ca2+, no uptake of calcium occurred and sperm motility and oxygen uptake were abolished. DISCUSSION The present dynamic studies using the more refined tracer technique confirm the static analyses o f Quinn and White (1966) and Karagiannidis (1976), which suggested a rise in the calcium concentration in ram and bull sperm following sudden cooling. The overall nett calcium concentration of the sperm is presumably dependent on the balance struck between calcium uptake b y the mitochondrial p u m p and its extrusion b y the plasma membrane p u m p (Bradley et al., 1979; Bradley and Forrester, 1980; Van Eerten and Forrester, 1980; Breitbart and Rubinstein, 1983; Breitbart et al., 1983, 1984). There is abundant evidence that cold shock causes p r o f o u n d permeability changes in sperm (Mann and Lutwak-Mann, 1955; Mayer, 1955; Blackshaw and Salisbury, 1957; Quinn and White, 1966; Tash and Mann, 1973; Karagiannidis, 1976). The electron microscope studies of Quinn et al. (1969) show considerable damage to the plasma membrane of ram sperm b u t much less change in the mitochondria on cold shocking. One effect of cold shock might therefore be to swing the calcium balance of the sperm at least temporarily in favour of a nett influx of calcium b y dislocating the outwardly directed plasma membrane pump, to a greater extent than the inwardly directed mitochondrial p u m p which m a y remain substantially intact. This suggestion is supported by the study of Van Eerten and Forrester ( 1 9 8 0 ) w h o f o u n d no further stimulation of calcium uptake in cold-shocked ram sperm on adding the membrane active antibiotic filipin, indicating that cold shock had already disrupted the plasma membrane. Peak calcium values were achieved 20--30 min after cold shocking and the subsequent decrease m a y be due to all membranes, including the mitochondrial, becoming " l e a k y " on incubation of the damaged sperm. Transmission electron microscope studies (Simpson et al., 1986) show extensive damage to, or removal of, plasma membranes from the head and middle piece of ram sperm cold shocked to 0°C. Sperm thawed after freezing in dry icealcohol to - 7 9 ° C t o o k up less calcium than those merely cold shocked at

140 0°C, presumably because the more severe treatment caused damage to the mitochondrial pump as well as the plasma membrane. As might be expected, calcium uptake increased as the temperature to which sperm were rapidly exposed was progressively reduced from 15°C to 0°C, indicating greater damage to the plasma membrane with the increase in temperature gradient. More surprising was the substantial increase in calcium levels on slow cooling to temperatures below 10°C. While it was not as great as after cooling sperm rapidly to these temperatures, the results indicate that the plasma membrane of ram sperm may suffer subtle irreversible damage even when precautions are taken to prevent cold shock. It is possible that at 10°C or below there m a y be a change in the phase of sperm membrane phospholipids. Evidence for such phase changes in h o m e o t h e r m s has been detected in rat liver mitochondria b y electron spin resonance (Raison et al., 1971a) and in bacteria by calorimetry (Steim et al., 1969). The absence of a phase change in the mitochondrial lipids of poikilotherms and temperature insensitive plants m a y be related to the high proportion of unsaturated fatty acids in their membranes (Raison et al., 1971b). Of particular interest in this respect are the observations of Holt and North (1984) that clustering of intramembranous particles over the tail of slowly cooled ram and blackbuck spermatozoa was only partially reversible on restoration of the physiological temperature. It is possible that the drastic rearrangement of membrane c o m p o n e n t s which occurs u p o n cooling below the phase transition temperature (Quinn, 1981) could also account for the increased membrane permeability which might in turn interfere with the motility and inevitably the fertility of the spermatozoa. The analyses of Karagiannidis (1976) did n o t detect any increase in calcium levels of bull spermatozoa rewarmed after slow cooling to 0°C and 5°C. This suggests that bull sperm m a y be less susceptible to low temperature damage than are ram sperm. On the other hand, it m a y merely reflect the greater sensitivity of the 4SCa2÷ tracer technique in detecting subtle damage to sperm under these conditions. Heating ram sperm at 50°C for 10 min irreversibly destroyed their motility and respiratory activity b u t the mechanism is clearly different from cold shock since heating had little effect on calcium uptake and large quantities of calcium were taken up if ram sperm were cold shocked after heating. Similar results have been obtained with bull sperm (Karagiannidis, 1976). R a m sperm, like bull and boar ( D o t t and Foster, 1975; Karagiannidis, 1976) can be temporarily immobilized b y a low concentration of formaldehyde. Motility can be restored b y dialysis and we have confirmed Karagiannidis' (1976) observation that sperm treated in this w a y are less susceptible to cold shock, as judged b y motility and oxygen uptake. This surprising p h e n o m e n o n m a y be due to stabilization of the plasma membranes since one of the effects of formaldehyde is to reduce the calcium uptake of the sperm.

141 T h e s p e r m i c i d a l p r o p e r t i e s o f d e t e r g e n t s have b e e n well d o c u m e n t e d ( K o e f o e d - J o h n s e n a n d M a n n , 1 9 5 4 ; C h o w et al., 1 9 8 0 ) . A d d i t i o n o f t h e d e t e r g e n t s , T r i t o n X - 1 0 0 , SDS a n d C T A B , r e n d e r e d t h e r a m s p e r m i m m o t i l e a n d r e s u l t e d in an i m m e d i a t e e f f l u x o f a c c u m u l a t e d c a l c i u m in c o n t r o l and c o l d - s h o c k e d sperm, d u e p r e s u m a b l y t o r u p t u r e o f s p e r m m e m b r a n e s . We c o n c l u d e t h a t t h e c a l c i u m levels o f r a m s p e r m are p e c u l i a r l y sensitive t o l o w e r i n g t h e e n v i r o n m e n t a l t e m p e r a t u r e b e l o w 15°C. The e f f e c t is aggrav a t e d if t h e rate o f c o o l i n g is rapid, a n d t h e resulting high c a l c i u m level, w h i c h m i g h t be d u e t o d i f f e r e n t i a l d a m a g e t o t h e p l a s m a and m i t o c h o n d r i a l m e m b r a n e p u m p s , c o u l d be a f a c t o r in c o l d shock. ACKNOWLEDGEMENT We are i n d e b t e d t o Miss K. M u r d o c h a n d Miss H. H u g h e s f o r their invaluable t e c h n i c a l resistance a n d t o t h e A u s t r a l i a n W o o l C o r p o r a t i o n f o r financial s u p p o r t a n d a p o s t - g r a d u a t e s t u d e n t s h i p (A.M.S.).

REFERENCES

Babcock, D.F., First, N.L. and Lardy, H.A., 1975. Transport mechanisms for succinate and phosphate localized at the plasma membrane of bovine spermatozoa. J. Biol. Chem., 250: 6488--6495. Babcock, D.F., First, N.L. and Lardy, H.A., 1976. Action of ionophore A23187 at the cellular level. Separation of effects at the plasma and mitochondrial membranes. J. Biol. Chem., 251: 3881--3886. Berridge, M.J., 1975. The interaction of cyclic nucleotides and calcium in the control of cellular activity. Adv. Cyclic Nucl. Res., 6: 1--98. Blackshaw, A.W., 1954a. The prevention of temperature shock of bull and ram semen. Aust. J, Biol. Sci., 7: 573--582. Blackshaw, A.W., 1954b. A bipolar electrode for the production of ejaculation in sheep. Aust. Vet. J., 30: 249--250. Blackshaw, A.W. and Salisbury, G.W., 1957. Factors influencing metabolic activity of bull spermatozoa in cold shock and its prevention. J. Dairy Sci., 40: 1099---1106. Bradley, M.P. and Forrester, I.T., 1980. A [Ca 2÷ + Mg2÷]-ATPase and active Ca 2÷ transport in the plasma membranes isolated from ram sperm flagella. Cell Calcium, 1: 381--390. Bradley, M.P. Van Eerten, M.T.W. and Forrester, I.T., 1979. The energy-dependent uptake of Ca 2÷ by mammalian spermatozoa, Proc. Univ. Otago Med. Sch., 57: 5--7. Breitbart, H. and Rubinstein, S., 1983. Calcium transport by bull spermatozoa plasma membranes. Biochim. Biophys. Acta, 732: 464--468. Breitbart, H., Stern, B. and Rubinstein, S., 1983. Calcium transport and Ca2*-ATPase activity in ram spermatozoa plasma membrane vesicles. Biochim. Biophys. Acta, 728: 349--355. Breitbart, H., Darshan, R. and Rubinstein, S., 1984. Evidence for the presence of ATPdependent calcium pump ATPase activities in bull sperm head membranes. Biochem. Biophys. Res. Commun., 122: 479--484. Brokaw, C.J., Josslin, R. and Bobrow, L., 1974. Calcium ion regulation of flagellar beat symmetry in reactivated sea urchin spermatozoa. Biochem. Biophys. Res. Commun., 58 : .795--800.

142 Chow, P.Y.W., Holland, M.K., Suter, D.A.I. and White, I.G., 1980. Evaluation of ten potential organic spermicides. Int. J. Fertil., 25: 281--286. Davis, B.K., 1978. Effect of calcium on motility and fertilization by rat spermatozoa in vitro. Proc. Soc. Exp. Biol. Med., 157 : 54--56. Dott, H.M. and Foster, G.C,, 1975. Preservation of differential staining of spermatozoa by formol citrate. J. Reprod. Fertil., 45: 47--60. Emmens, C.W., 1947. The motility and viability of rabbit spermatozoa at different hydrogen-ion concentrations. J. Physiol. Lond., 106: 471--481. Garbers, D.L. and Kopf, G.S., 1980. The regulation of spermatozoa by calcium and cyclic nucleotides. Adv. Cyclic Nucl. Res., 13: 252--306. Glover, T.E. and Watson, P.F., 1985. Cold shock and its prevention by egg yolk in spermatozoa of the cat (Fells catus). Cryo-letters, 6: 239--244. Green, D.P.L., 1978. The induction of the acrosome reaction in guinea-pig sperm by the divalent metal cation ionophore A23187. J. Cell. Sci., 32 : 37--151. Holt, W.V. and North, R.D., 1984. Partially irreversible cold-induced lipid phase transitions in mammalian sperm plasma membrane domains: freeze-fracture study. J. Exp. Zool., 230: 473--483. Hyne, R.V. and Garbers, D.L., 1979a. Calcium-dependent increase in adenosine 3',5'monophosphate and induction of the acrosome reaction in guinea pig spermatozoa. Proc. Natl. Acad. Sci. U.S.A., 76: 5699--5703. Hyne, R.V., Garbers, D.L., 1979b. Regulation of guinea pig sperm adenylate cyclase by calcium. Biol. Reprod., 21: 1135--1142. Jamil, K. and White, I.G., 1981. Induction of acrosomal reaction in sperm with ionophore A23187 and calcium. Axchiv. Androl., 7 : 283--292. Karagiannidis, A., 1976. The distribution of calcium in bovine spermatozoa and seminal plasma in relation to cold shock. J. Reprod. Fertil., 46: 83--90. Koefoed-Johnsen, H.H. and Mann, T., 1954. Studies on the metabolism of semen. Effect of surface active agents with special reference to the oxidation of succinate by spermatozoa. Biochem. J., 57 : 406--410. Kopf, G.S. and Garbers, D.L., 1980, Calcium and a fucose-sulphate rich polymer regulate sperm cyclic nucleotide metabolism and the acrosome reaction. Biol. Reprod., 22: 1118--1126. Lehninger, A.L., Carafoli, E. and Rossi, C.S., 1969. Energy-linked ion movements in mitochondrial systems. Adv. Enzymol., 29: 259--320. MeGrady, A.V., Nelson, L. and Ireland, M., 1974. Ionic effects on the motility of bull and chimpanzee spermatozoa. J. Reprod. Fertil., 40:71--76. Mann, T. and Lutwak-Mann, C., 1955. Biochemical changes underlying the phenomenon of cold shock in spermatozoa. Arch. Sci. Biol., 39: 578--588. Mayer, D.T., 1955. The chemistry and certain aspects of the metabolic activities of mammalian spermatozoa. Mich. Univ. Cent. Syrup. Reprod. and Infertil., Michigan State University, East Lansing, pp. 45--53. Morton, B., Harrigan, J., Albagli, L. and Jooss, T., 1973, The activation of motility in quiescent hamster sperm ifrom the epididymis. Biol. Reprod., 9: 71--72. Peterson, R.M., Russell, L., Bundman, D. and Freund, M., 1978. Effect of ionophore induced calcium uptake and dibutrylcyclic AMP on the acrosome membrane of boar spermatozoa. Proc. Fed. Am. Soc. Exp. Biol., 37 : 380. Peterson, R.M., Seyler, D., Bundman, D. and Freund, M., 1979. The effect of theophylline and dibutryl cyclic AMP on the uptake of radioactive calcium and phosphate ions by boar and h u m a n spermatozoa. J. Reprod. Fertil., 55: 385--390. Quinn, P.J., 1981. The fluidity of cell membranes and its regulation. Prog. Biophys. Mol. Biol., 38: 1--104. Quinn, P.J. and White, I.G., 1966. The effect of cold shock and deep-freezing on the concentration of major cations in spermatozoa. J. Reprod. Fertil., 12: 263--270.

143

Quinn, P.J., White, I.G. and Cleland, K.W., 1969. Chemical and ultrastructural changes in ram spermatozoa after washing, cold shock and freezing. J. R,eprod. Fertil., 18: 209--220. Quinn, P.J., White, I.G. and Voglmayr, J.K., 1970. Use of a continuous flow dialysis apparatus to determine the effect of cations, buffers and osmotic pressure on ram spermatozoa. J. Reprod. Fertil., 22 : 253--260. Raison, J.K., Lyons, J.M., Melhorn, R.J. and Keith, A.D,, 1971a. Temperature-induced phase changes in mitochondrial membranes detected by spin labeling. J. Biol. Chem., 246: 4036--4040. Raison, J.K., Lyons, J.M. and Thomson, W.W., 1971b. The influence of membranes on the temperature-induced changes in the kinetics of some respiratory enzymes of mitochondria. Arch. Biochem. Biophys., 142: 83--90. Rosada, A., Hicks, J.J., Martinez-Zedillo, G., Bondani, A. and Martinez-Manatou, J., 1970. Inhibition of human sperm motility by calcium and zinc ions. Contraception, 2: 259--273. Shams-Borhan, G. and Harrison, R.A.P., 1981. Production, characterization and use of ionophore-induced calcium-dependent acrosome reaction in ram sperm. Gamete Res., 4: 407--432. Simpson, A.M., Swan, M.A. and White, I.G., 1986. Action of phosphatidylcholine in protecting ram sperm from cold shock. Gamete Res. (in press). Steim, J.M,, Tourtellotte, M.E., Reinert, J.C., McElhaney, R.N. and Rader, R.L., 1969. Calorimetric evidence for the liquid-crystalline state of lipids in a biomembrane. Proc, Natl. Acad. Sci. U.S.A., 63: 104--109. Tash, J.S. and Mann, T., 1973. Adenosine 3'-5'-cyclic monophosphate in relation to motility and senescence of spermatozoa. Proc. R. Soc. Lond. B., 184: 109--114. Van Eerten, M.T.W. and Forrester, I.T., 1980. Biochemical aspects of ram sperm viability. Proc. N.Z. Soc. Anim. Prod., 40: 130--135. Van Eerten, M.T.W., Bradley, M.P. and Forrester, I.T., 1978. A new method for estimation of sperm viability. Proc. Univ. Otago Med. Sch., 56: 104--105. Wales, R.G. and White, I.G., 1959. The susceptibility of spermatozoa to temperature shock. J. Endocrinol,, 19: 211--220.