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Nucleotide specificity of the “ATP-dependent” surface reaction of the acrosomal region of the bull sperm head
Printed in Sweden Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any form resewed
Experimental Cell Research 87 (1974) 47-54
NUCLEOTIDE
SPECIFICITY
SURFACE REACTION
OF THE “ATP-DEPENDENT”
OF THE ACROSOMAL
REGION
OF THE
BULL SPERM HEAD P. E. LINDAHL Institute of Zoophysiology, University of Uppsala, 75122 Uppsala I, Sweden
SUMMARY In the presence of 2 x 10m6M Ca2+ not only ATP, but also ITP, GTP, UTP and CTP, induce head-to-head association in washed bull spermatozoa (table 1). The low substrate specificity is thus similar to that of purified actomyosin. As only addition GTP, ITP, UTP and CTP are able to induce association, i.e. at the low concentration of activators of the medium (table 2). However, in a further experiment of this kind CTP failed in this respect, suggesting a need for higher concentration of activator with some samples of spermatozoa. ADP as only addition produced rather varying results; out of 9 experiments one induced decreased and 4 induced increased association, whereas 4 had no significant effect. In the case of ADP Mg2+ was more effective as an activator than Ca2+, which is the more effective activator of ATP. The reason for this difference is discussed.
When brought into contact with exogenous ATP in presenceof Mg2+ or Ca2+,mammalian spermatozoa associate with their acrosomal regions [8, IO]. This reaction is blocked by salyrgan, “one of the most specific inhibitors of the contractile proteins of actomyosin type” [2], and by ethylene diamine tetraacetic acid (EDTA), and it has been suggested that a Mg2+- or Ca2+-activated ATPase, located at the outside of the cell membrane, is involved in the reaction [8, lo]. According to the views of modern colloid science, reduction of the surface charge may lead to association of cells (cf [S]). In connection with a hypothesis advanced by B. M. Jones [5] and P. C. T. Jones [6] it was proposed that a decrease in surface charge is brought
about
in mammalian spermatozoa by a detachment of negatively charged groups from the sur4-741816
face by contraction of an actomyosin complex in the surface, or by some other conformational change induced by the splitting of ATP. It is the aim of the present investigation to study and compare the effects of a number of nucleoside triphosphates on the kind of association mentioned. Special attention was further paid to the effect of ADP, the only nucleoside diphosphate studied. MATERIAL
AND METHODS
Semen was obtained as second ejaculates from bulls of the Swedish White and Red Breed with sperm pictures which were normal, although the bulls showed chromosome abnormalities in meiosis and were sterile. The semen was slowly cooled to room temperature, about 22”C, part of it being used about 1 h after collection. The rest was cooled further to 5”C, stored at this temperature, and used at room temperature 25 h after collection. As media in the experiments and for washing of Exptl Cell Res 87 (1974)
48
P. E. LindaId
the spermatozoa Tris-HCl-buffered or phosphatebuffered (for further details, cf [lo]) solutions of pH 7.40 and 7.35, respectively, were used. The former, 0.050 M with regard to Tris (hydroxymethyl)-aminomethane and 0.041 M with regard to HCI, was made slightly hypertonic by addition of 0.218 M raffinose (cf [IO]). This medium is 2 b’ IO-” M and 4 lO-7 M with regard to Ca”+ and Mg”+, respectively (analyses performed by the Analytical Department of the Institute of Chemistry, University of Uppsala). The other solution, being slightly hypotonic, consisted of 0.071 M phosphate buffer containing 6.00 g glucose and 2.00 g galactose per liter. The activators, Ca”+ or Mg’+, were dissolved in the medium in appropriate concentrations, as were all the nucleotides to be studied. All nucleotides studied were freed from divalent cations by application of an appropriate amount of the sodium salts to an IRC-50 (H+) column and elution with distilled water. The eluates were adjusted to the pH of the actual medium by diluting them 10 times with the medium to be used (cf [IO@. The spermatozoa were washed three times by centrifugation (600 g for 10 min) and were resuspended in the medium, each time at approx. 20 vol per original volume of semen. The final sperm density was adjusted to about 40000 cells/PI. Each suspension of washed spermatozoa, stored at room temperature, was used for 6 h only, during which period a slight increase in the frequency of association took place. Occasionally this increase was more marked, and in such cases a new suspension was prepared. The degree of association of the spermatozoa in a suspension is determined by counting the number of associated cells and the total number of cells in a specified volume. Changes in the frequency of association caused by additions to a sperm suspension are established in the following way [ll]. From the percentage of associated sperms, P1, in a test sample and that of Pz, in a blank sample the difference, DPz -I’,, is calculated. D :,O means that associated cells have become dissociated. D 10 means that the substance or combinations of substances tested have induced association. Each determination, giving a mean difference, 4, comprises 10 or more such pairs of counts. The suspensions from which the samples are taken are composed of (I) 4 vol medium; of which 1 to 4 may be exchanged for medium containing substances to be tested; (2) 1 vol sperm suspension. The sperm suspension is always added last. Five minutes are allowed for the substances to act and for the spermatozoa to sediment in the hemocytometer before the counting is started. The two countings of each pair are completed within about 17 min after the mixing of sample and blank. Students t-test is applied in order to establish whether the mean differences found deviate significantly from zero. A quantitative comparison between the degree of association or dissociation induced by different substances or combinations of substances is generally possible only when the pairs of counts to be compared have been performed in regular alternation with use of the same suspension or suspensions of spermatozoa. The chemical products used originate from the folExptl Cell Res 87 (1974)
lowing sources. All nucleotides were pur-chased from Sigma Chemical Co, USA, viz., adenosine 5’-diphosphate, Sigma No. A-01271 adenosine 5’-triphosohate. Sigma No. A-3127: cvtidine 5’-triohosnhate. Sigma No. C-1506; guano& 5’-triphosphate, Sigma No. G-8502: inosine 5’-triohosohate. Sigma No. l5000; Uridine 5’-triphosphate, ‘Sigma No. U-6500: further Tris-(hydroxymethyl)aminomethane. Sigma 7-9 (T 1378) Sigma Chemical Co, USA; raffinose (Pentahydrat), Merck, Germany. All other chemicals were of analytical grade. All solutions were made up with water distilled twice in a quartz apparatus.
RESULTS As is shown in table 1, three of the four studied nucleoside triphosphates induced increased frequency of association when added without activator to washed spermatozoa in the Tris-HCl buffered medium. The deviating nucleotide was CTP. In a second series of experiments of the same kind all four nucleoside triphosphates significantly increased the association. Further addition of CaZ+ caused association to increase significantly in all 4 cases. The effects of the different nucleoside triphosphates did not differ significantly from that of ATP (table 2). ADP was studied in similar experiments to those of table 1, although in phosphate buffered medium. When added without activator to the spermatozoa, ADP decreased association in one experiment and increased it in 4 experiments out of nine (table 3). In 4 experiments there was no significant change. When additions of Mg2+ or Ca2+ in final cont. of 2 x 10e6, 7 x 10~~ or 2 x 10-j M were also made, the activators significantly increased association except in the case of 2 % 1O-6 M Mg2+ (table 3). It should be observed that the final concentration of ADP in the actual experiments was 2 x 1O-4 M. The experiments of table 4 indicate that the effects of ADP shown in table 3 appeared also in the absence of phosphate at 10 times lower concentration of ADP. The same ef-
Nucleotide specificity of head-to-head association
49
Table 1. Effects of some nucleoside triphosphdtes aloize and in combination with Cazf on the frequency of association in washed bull spermatozoa in Tris-HCI-buffered
medium
Final concentrations of both nucleoside triphosphates and Ca 2+2 x 1O-5M. For the UTP-experiment 2 suspensions from one ejaculate and one suspension from another ejaculate were used; for the other experiments 2 suspensions from one ejaculate were used
Additions Sample 1 2 3 : 3 1 2 3 1 :
None CTP CTP + Ca2+ None UTP UTP + Ca2+ None GTP GTP -1Ca2+ None ITP ITP + Caz+
Mean difference 4f,-Z,iS.E. -0.08kO.26
Deviation of q-2, from zero t
P
Mean difference &,,kS.E.
t
2.30
Deviation of &-2,-W, from zero
Effect of Ca2+, &--2)-W, +S.E.
P
0.31
t
+ 1.7110.30 - 1.79kO.24
-0.4610.20
Deviation of h-3, from zero
7.46
P
5.70
~0.05
+ 1.84kO.24 7.67
i- 1.3510.28
4.82
+ 1.38i0.22
6.27
-2.31 kO.23 10.04 < 0.001 1 -0.38kO.17 -0.65kO.19
2.24 3.42
-0.05 - 1.73 kO.34
5.09
- 2.03 iO.23
8.83
co.01
feet of ADP was exerted when the medium consisted chiefly of seminal plasma (table 5). It appears from table 6 that Mg2+ was a more efficient activator than Ca2+when ADP increased the frequency of association.
DISCUSSION In the presence of Ca2+ all of the studied nucleoside triphosphates, ATP, CTP, ITP, GTP, and UTP, induce increased head-to-
Table 2. Comparison between the potencies of some nucleoside triphosphates in inducing headto-head association in washed bull spermatozoa in presence of Ca2+ The effect of ATP is used as a standard with which the effects of the other nucleotides are compared. This is done in two different experiments with different spermatozoa. Final concentrations of nucleotides and Ca2+ 2 x 1O-5 M Deviation of B from zero Sample
Additions
Mean difference, ii&S.E.
1 2 3 4
None ATP + Ca2+ CTP + Ca2+ UTP + Ca2+
O_,1-3,= -0.96kO.24 D,,-,, = -0.70+0.14
: 3 4
None ATP + Ca2+ GTP + Ca2+ ITP +Ca2+
<,I-2, = -0.8210.15
i&,,
= -0.45f0.10
Q--3) = -0.38 )0.09 D+-4, = -0.56&0.10
t
P
5.47
Mean diff. between effect of ATP and that of other NTP 4kS.E.
&-,1-3,= 0_(1+,1-4,=
-0.14kO.21 +0.13+0.18
D,,-,,-,,-a,=
-0.27t0.20
Deviation of differences from zero
t
P
0.67 0.72 1.05
4.57
@(1-2,--(1--3) = +0.07f0.10
0.70
5.6OiO.001
l&+-(1--4) = -0.12kO.16 D,~--3,--(1-~~= +0.19+0.17
0.75
1.12
Exptl Cell Res 87 (1974)
50
P. E. Lindahl
Table 3. Effect of ADP alone and in combination Iz’ith Ca’i or Mg:“+ 011 the jr.cylrcvlr:l. of association of thrice-washed bull spermatozoa in phosphate-buffered medium In all cases when ADP is the only addition the total concentration of Ca” is 3 IO-” M, and that of Mg” probably lower. One or two ejaculates were used in each experiment consisting of one or two tests. } marks components acting simultaneously
Expt no.
Addition
Added concentration (M)
ADP ADP ADP ADP ADP Mg*+ ADP ADP Ca2+ I ADP ADP Mg2+ ADP ADP Ca2+ ADP ADP Mg2+ ADP ADP Ca2+
2 A IO-” 2 _ 10-4 2; IO-” 2 /~IO-” 2 Y 1o-4 2 I: IO-6 2 >:IO-4 2 _’ IO-” 2 - 10-e 2 :
1
5 6
1 1 1 1
I
8 9
Mean difference D-tS.E.
Deviation of d from zero
Significance of effect of addition of activator
t
4kS.E.
P
1.2OkO.29 0.77 t-o.37 0.33 iO.20 -0.55+0.19 m-0.54&0.25
4.14 2.08 1.65 2.18 2.16
-0.18kO.20 -1.34-to.41
3.27 0.90
~=o.ol
em0.15+0.29 ~ 2.05 -to.49
4.18 0.52
-rO.Ol
-0.37kO.14 -- 1.00+0.22
4.55 2.64
:m0.46f0.21 1.61kO.26 - 1.13iO.25 -2.56kO.63
t
P
-.O.Ol (< - 0.02 0.05)
0.01 kO.28
0.04
- 1.16iO.41
2.83
‘. 0.02
~ 1.90+0.40
4.75
- 0.001
co.01 < 0.05 I
-0.63 kO.19
3.32
-0.01
2.19 6.19
-< 0.05 0.001
- 2.07 + 0.23
9.00
4.52 4.06
-z0.01 < 0.01
- 1.43 20.65
2.20
head association (table 1). The substrate specificity is thus rather low and independent of whether the base of the nucleoside triphos-
> 1
I
0.001 ==0.05
phate is a purine or a pyrimidine. The reason for using Ca2+ as activator, instead of Mg2+, was the fact that Ca2+ has turned out to be
Table 4. Effect of ADP alone or in combination with Ca2+ on the frequency of association of thrice-washed bull spermatozoa in Tris-HCI buffered medium One ejaculate was used in each experiment. } marks components present simultaneously
Expt no.
1 2 3
Addition
i { {
ADP ADP Ca2+ ADP ADP Ca2+ ADP ADP Ca2+
Concentration (M)
Deviation of 4 from zero Mean difference t D f S.E.
1 1 2 s10-5 1 2 X 10-S 2 x 10-b 2 x 10-S 2 x 10-S 2 x 10-h 2 x 10-E 2 x 10-S 2 x 10-s
Exptl Cell Res 87 (1974)
Significance of Ca”+-effect
P
-0.52kO.24
2.17
J 0.05
- 1.61iO.21 + 0.07 * 0.22
7.67 0.32
< 0.001
-0.68f0.15 -0.7220.22
4.53 3.27
co.01 co.01
- 1.31 kO.28
4.68
< 0.001
3 J
DkS.E.
t
P
- 1.09+ 0.24
4.54
‘- 0.01
-0.75+0.19
3.95
.-0.01
-0.59kO.17
3.47
‘--0.01
Nucleotide specificity of head- to-head association Table 5. Effect of ADP alone on the frequency of association of bull spermatozoa in 4 parts of seminal plasma and one part of phosphatebuffered medium One ejaculate was used in each experiment
Expt no. 1 2
Deviation of Concentra4 from zero tion of ADP Mean difference 4kS.E. t P CM) 2 x 10-b 2 x 10-b
-0.50+0.15 -0.33kO.09
3.33 3.65
-z0.01 < 0.01
more efficient in studies with ATP [2]. Similar properties have been demonstrated for purified actomyosin [l, 7, 16, 18, 201. When ATP is supplied as only addition to washed spermatozoa in phosphate or TrisHCl buffered medium, no association is induced [9]. As ATP does induce association when Mg2+ or Ca2+ is also added, the fact that this process failed to appear without addition of activators was looked upon as resulting from too low a concentration of the two activators in the media used. All the studied nucleoside triphosphates were able to induce association when added alone; although, for CTP this was true only in one case out of two. We thus find that ATP and,
51
although to a lesser degree, also CTP, in which compounds the 6-position in the purine and pyrimidine rings, respectively, is substituted with NH,-, need a higher concentration of Ca2+ than those in which this position is substituted with OH-. A corresponding grouping of nucleoside triphosphates appears in studies by Hasselbath [4] of contraction of actomyosin gel and simultaneous hydrolyses of the triphosphates into corresponding diphosphates and Pi. According to this author the developed tension was large, the rate of hydrolyses high, and the need for Mg2+ little with ATP and CTP, whereas with UTP, ITP, and GTP tension was small, the rate of hydrolyses high, and the need for Mg2+ great. The rate of hydrolyses of the different nucleoside triphosphates increased with increasing concentration of Mg2+ until an optimum concentration was reached. At intermediate concentrations of Mg2+ the rates of hydrolyses form a series of decreasing values from ATP to GTP in the order ATP>CTP>UTP> ITP > GTP [4]. A corresponding variation in the capacity of the different nucleoside triphosphates to induce head-to-head association at a certain concentration of Ca2+ was not observed (table 2).
Table 6. Comparison between the abilities of A4g2+ and Ca2f to induce head-to-head association in the presence of ADP Medium based on Tris-HC1 buffer. Spermatozoa from one ejaculate used in each experiment
Expt no.
Additions ADP
1
Mg2+ t ADP Ca2+ ADP
2
1 ADP Mg2+ Ca2+
Concentra- Mean tion difference ii+S.E. CM) 2x10-4 x 1 IO-5 21 x 10-s 10-a 1 x 10-S X 10-s 11 x 10-S 1 x 10-S
Deviation from zero
_
-
Deviation from zero
D~gz+-D&+
t
P
-1.18iO.22
5.36
-0.72kO.17
4.24
< 0.01
-0.9lkO.19
4.79
< 0.001
-0.42+0.11
3.82
< 0.01
*SE.
t
P
+0.46?0.17
2.71
< 0.05
+0.4810.10
4.80
< 0.001
Exptl Cell Res 87 (1974)
52
P. E. Linda111
The need for a higher concentration of CaZ+ in the cases of ATP and CTP than in the cases of ITP, GTP and UTP may depend on a similar structure of the active centre in the ATPase of the acrosome covering membrane to that suggested for myosin ATPase. The conception of the mentioned structure was based on studies of the interaction between myosin and its substrates under the influence of different reagents (modifiers; for review see Perry [ 151p. 481). Substances containing enone structure, among others the pyrimidines cytosine and uracil and the nucleoside guanosine, were found to block the “ATP-dependent” reaction [9]. This property seems to be suppressed in some way when the corresponding nucleoside triphosphates function as substrates. In a previous study [8] ADP was found to induce decrease in the frequency of association in twice washed spermatozoa. When this experiment was repeated some years later in the same manner, there was an increase in the frequency of association. It thus seemed necessary to study the effect of ADP on a broader scale and with varied concentration of Mg2+ and Ca2+ (table 3). Decreased frequency of association was effected only in one case out of 9 by ADP alone, indicating that this is a possible but rare consequence of this treatment. Obviously there is a great variability between different sperm samples, as at this low concentration of activators (cf p. 48) no significant effect appeared in 4 cases, while increased association appeared in 4 cases. In this respect ADP differs from ATP. It should, however, be stressed that the concentration of ADP in the present experiments was 10 times higher than that in the corresponding previous experiments with ATP [lo]. With one exception, addition of low concentrations of Mg2+ and Ca2+ to samples containing ADP induced further increase in Exptl Cell Res 87 (1974)
association (table 3). In order to rule out the possibility that the relatively high concentration of ADP and the buffer system used, cooperated in or contributed to the increased association appearing in table 3, a few similar experiments were performed with a 10 times lower concentration of ADP and the Tris-HC1 buffered medium (table 4). The results were rather similar to those in table 3, indicating that neither the higher concentration of ADP nor of the phosphate buffer are of any importance for the observed effect of ADP. The original experiments with ADP, referred to [8], were performed with spermatozoa washed only twice. The concentrations of seminal plasma components might have been higher in those experiments than in the present ones. Therefore the effect of ADP as only addition to seminal plasma, diluted 4 parts to one part of phosphate-buffered medium (table 5) was studied. It should be pointed out that seminal plasma contains Mg2+ and Ca2+ in concentrations high enough to permit these ions to function as activators of the ATP-dependent reaction (cf [IO] p. 428). The effect of ADP was just the same in the presence of seminal plasma components (table 5) as in their absence (table 3) and these do not seem to have any special influence. The comparatively higher effect of Mg’-1 than of Ca2+ in pairs 6-7 and 8-9 of experiments referred to in table 3 suggests that Mg2+ is more efficient than Ca2+ in causing head-to-head association in the presence of ADP. Experiments in which both ions were tested with spermatozoa from the same suspension confirm this suggestion (table 6). As the hypothesis upon which the present investigations originally were based implies that head-to-head association is connected with hydrolyses of ATP, the present results should be discussed against this background. Some different possibilities concerning the
Nucleotide specificity of head-to-head association enzymic splitting of ATP, may be discussed. (1) ADP is hydrolyzed by the same enzyme as ATP. If this is the case it is remarkable that Mg2+ is a more efficient activator than Ca2+, whereas this is reversed when ATP functions as the substrate. These facts might possibly depend on the relevant stability constants for the two nucleotides and the cations in question. However, inspection of the constants [19] reveal relationships suggesting that the most efficient activator should be Mg2+ for ATP and Ca2+ for ADP, i.e., just the opposite to what was found in the experiments. (2) ADP is transformed into ATP and AMP by adenylate kinase. If this reaction is slower than the splitting of ATP, it will determine the rate of hydrolyses of ATP when ADP is the only exogenous substrate. As a matter of fact, Mg2+ is more efficient than Ca2+ as activator of adenylate kinase from most tissues, although the reverse has also been observed [13]. This manner of reasoning implies that the adenylate kinase should be on the outside of the acrosome covering membrane (cf below). (3) ADP acts in quite a different way, e.g. by increasing the permeability for ATP in the mentioned membrane, thus releasing ATP (and activators) from the interior of the cell. Because of the relatively fast action of exogenous ATP on head-to-head association it was inferred that this slowly penetrating substance must act on the outer surface of the cell membrane [lo]. This conclusion should be extended to other nucleoside triphosphates and most probably also to ADP. Thus, when scrutinizing the literature for enzymes hydrolyzing nucleoside triphosphates and ADP, only statements referring to intact spermatozoa are of interest for the current problem. Further, we confine ourselves to
53
information concerning Mg2+- or/and Ca2+activated ATPase, as further addition of Na++K+ had no effect on head-to-head association that could not be related to increased ionic strength [lo]. The above-mentioned conditions are fulfilled by experiments with spermatozoa from three species, viz., bull [14, 171, ram [17, 211 and man [3]. In all cases the ATPase was activated by Mg2+. Ca2+ was also effective in bull [3] and ram [21] spermatozoa, in the latter study more so than Mg2+. ADP was hydrolysed by bull [14], ram [21] and human [14] spermatozoa. According to Vogelmayr et al. [21] negligible amounts of AMP were found when both ATP and ADP were present at the same time, “indicating a preferential hydrolysis of ATP”. With human spermatozoa [3] 80% of the added ATP was hydrolysed (37°C) after 20 min and about twice as much ADP as AMP was present. CTP and ITP were hydrolysed by bull spermatozoa [14] and, by human spermatozoa, AMP too was dephosphorylated
131. Observations made with spermatozoa of one species may not be generalized to apply to all three species, and can only be stated tentatively. If the acrosome covering membrane has the same enzymic outfit as the rest of the surface membrane then the interpretation of the present observations on head-tohead association, as resulting from enzymic splitting of certain substrates, is well in keeping with the facts reported above from other investigations. Nelson [12] proposed the occurrence of adenylate kinase in bull spermatozoa, and this would provide an easy explanation for the results [3, 14, 211 accounted for above (cf [14]). To my knowledge, this enzyme has still not been definitely demonstrated in mammal spermatozoa. It is nevertheless of interest to note that the dephosphorylation Exptl Cell Res 87 (1974)
54
P. E. Linduhl
rates, when ADP
is the only substrate, are 209); lower than with ATP alone [21]. Such a relationship was assumed as an explanation of the fact that Mg”+mwas a more efficient activator than Ca2+ when the association was induced by ADP (p. 53). The great variation in the results obtained with ADP as only addition to washed spermatozoa may possibly have some relationship to the degree of maturation or senescence of the spermatozoa in different ejaculates. This would mean that these processes lead to changes in the permeability of divalent cations in the part of the cell membrane in question. 1 am indebted to Dr T. Svensson, former Chief Veterinarian of the AI-Station at Enkoping, and to Dr B. Gustafsson. Head of the Clinic for Obstetrics and Gynaecology,‘the Veterinary College, Stockholm, for generous supply of bull semen. I thank Mrs Gun Ronnqvist for very efficient assistance. This investigation was supported by the Swedish Natural Science Research Council.
Bettex-Galland, M & Ltisher, E F. Advances in protein them 20 (1965) 1. 3. Durr, I F, Abla, A & Mroueh, A. J reprod fert 31 (1972) 313. 4. Hasselbach. W. Biochim bioohvs - acta 20 (1956) 355. 5. Jones, B M. Nature 212 (1966) 362. 6. Jones, P C T, Nature 212 (1966) 365. 7. Kiellev, W W & Blum. J J. Arch biochem biophys 55 (1955) 486. 8. Lindahl, P E, Exptl cell res 53 (I 968) 626. 9. -- Ibid 53 (1968) 639. 10. - Ibid 81 (1973) 413. II. Lindahl, P E, Bodin, N 0 & Brattsand, R, lnt j fert 8 (1963) 825. 12. Nelson, L, J cell physiol 68 (1966) 113. 13. Noda, L, The enzymes (ed P D Boyer) vol. 8, p. 279. Academic Press, New York (1973). 14. O’Donnell, J M & Ellory, J X, Biochem sot (London), agenda papers 8-9 June 1972, p. 17. 15. Perry, S V, Ann rev biochem 30 (1961) 473. 16. Portzehl, H, Biochim biophys acta 14 (1954) 195. 17. Quinn, P J & White, I G, J reprod fert 15 (1968) 449. 18. Ranney, R E, Am j physiol 178 (1954) 517. 19. Sillen, L G & Martell, A (compilers), Stability constants of metal ion complexes. 2nd edn. Special pub1 No 17. The Chemical Society, London (1964). 20. Spicer, S S & Bowen, W J, J biol them 188 (1951) 741. 21. Vogelmayr, J K, Quinn, P J & White, I G, Biol reprod 1 (1969) 215. 2.
REFERENCES 1. Bergkvist, R & Deutsch, A, Acta them Stand 8 (1954) 1105.
Exptl Cell Res 87 (1974)
Received February 1, 1974
Experimental Cell Research 87 (1974) 47-54
NUCLEOTIDE
SPECIFICITY
SURFACE REACTION
OF THE “ATP-DEPENDENT”
OF THE ACROSOMAL
REGION
OF THE
BULL SPERM HEAD P. E. LINDAHL Institute of Zoophysiology, University of Uppsala, 75122 Uppsala I, Sweden
SUMMARY In the presence of 2 x 10m6M Ca2+ not only ATP, but also ITP, GTP, UTP and CTP, induce head-to-head association in washed bull spermatozoa (table 1). The low substrate specificity is thus similar to that of purified actomyosin. As only addition GTP, ITP, UTP and CTP are able to induce association, i.e. at the low concentration of activators of the medium (table 2). However, in a further experiment of this kind CTP failed in this respect, suggesting a need for higher concentration of activator with some samples of spermatozoa. ADP as only addition produced rather varying results; out of 9 experiments one induced decreased and 4 induced increased association, whereas 4 had no significant effect. In the case of ADP Mg2+ was more effective as an activator than Ca2+, which is the more effective activator of ATP. The reason for this difference is discussed.
When brought into contact with exogenous ATP in presenceof Mg2+ or Ca2+,mammalian spermatozoa associate with their acrosomal regions [8, IO]. This reaction is blocked by salyrgan, “one of the most specific inhibitors of the contractile proteins of actomyosin type” [2], and by ethylene diamine tetraacetic acid (EDTA), and it has been suggested that a Mg2+- or Ca2+-activated ATPase, located at the outside of the cell membrane, is involved in the reaction [8, lo]. According to the views of modern colloid science, reduction of the surface charge may lead to association of cells (cf [S]). In connection with a hypothesis advanced by B. M. Jones [5] and P. C. T. Jones [6] it was proposed that a decrease in surface charge is brought
about
in mammalian spermatozoa by a detachment of negatively charged groups from the sur4-741816
face by contraction of an actomyosin complex in the surface, or by some other conformational change induced by the splitting of ATP. It is the aim of the present investigation to study and compare the effects of a number of nucleoside triphosphates on the kind of association mentioned. Special attention was further paid to the effect of ADP, the only nucleoside diphosphate studied. MATERIAL
AND METHODS
Semen was obtained as second ejaculates from bulls of the Swedish White and Red Breed with sperm pictures which were normal, although the bulls showed chromosome abnormalities in meiosis and were sterile. The semen was slowly cooled to room temperature, about 22”C, part of it being used about 1 h after collection. The rest was cooled further to 5”C, stored at this temperature, and used at room temperature 25 h after collection. As media in the experiments and for washing of Exptl Cell Res 87 (1974)
48
P. E. LindaId
the spermatozoa Tris-HCl-buffered or phosphatebuffered (for further details, cf [lo]) solutions of pH 7.40 and 7.35, respectively, were used. The former, 0.050 M with regard to Tris (hydroxymethyl)-aminomethane and 0.041 M with regard to HCI, was made slightly hypertonic by addition of 0.218 M raffinose (cf [IO]). This medium is 2 b’ IO-” M and 4 lO-7 M with regard to Ca”+ and Mg”+, respectively (analyses performed by the Analytical Department of the Institute of Chemistry, University of Uppsala). The other solution, being slightly hypotonic, consisted of 0.071 M phosphate buffer containing 6.00 g glucose and 2.00 g galactose per liter. The activators, Ca”+ or Mg’+, were dissolved in the medium in appropriate concentrations, as were all the nucleotides to be studied. All nucleotides studied were freed from divalent cations by application of an appropriate amount of the sodium salts to an IRC-50 (H+) column and elution with distilled water. The eluates were adjusted to the pH of the actual medium by diluting them 10 times with the medium to be used (cf [IO@. The spermatozoa were washed three times by centrifugation (600 g for 10 min) and were resuspended in the medium, each time at approx. 20 vol per original volume of semen. The final sperm density was adjusted to about 40000 cells/PI. Each suspension of washed spermatozoa, stored at room temperature, was used for 6 h only, during which period a slight increase in the frequency of association took place. Occasionally this increase was more marked, and in such cases a new suspension was prepared. The degree of association of the spermatozoa in a suspension is determined by counting the number of associated cells and the total number of cells in a specified volume. Changes in the frequency of association caused by additions to a sperm suspension are established in the following way [ll]. From the percentage of associated sperms, P1, in a test sample and that of Pz, in a blank sample the difference, DPz -I’,, is calculated. D :,O means that associated cells have become dissociated. D 10 means that the substance or combinations of substances tested have induced association. Each determination, giving a mean difference, 4, comprises 10 or more such pairs of counts. The suspensions from which the samples are taken are composed of (I) 4 vol medium; of which 1 to 4 may be exchanged for medium containing substances to be tested; (2) 1 vol sperm suspension. The sperm suspension is always added last. Five minutes are allowed for the substances to act and for the spermatozoa to sediment in the hemocytometer before the counting is started. The two countings of each pair are completed within about 17 min after the mixing of sample and blank. Students t-test is applied in order to establish whether the mean differences found deviate significantly from zero. A quantitative comparison between the degree of association or dissociation induced by different substances or combinations of substances is generally possible only when the pairs of counts to be compared have been performed in regular alternation with use of the same suspension or suspensions of spermatozoa. The chemical products used originate from the folExptl Cell Res 87 (1974)
lowing sources. All nucleotides were pur-chased from Sigma Chemical Co, USA, viz., adenosine 5’-diphosphate, Sigma No. A-01271 adenosine 5’-triphosohate. Sigma No. A-3127: cvtidine 5’-triohosnhate. Sigma No. C-1506; guano& 5’-triphosphate, Sigma No. G-8502: inosine 5’-triohosohate. Sigma No. l5000; Uridine 5’-triphosphate, ‘Sigma No. U-6500: further Tris-(hydroxymethyl)aminomethane. Sigma 7-9 (T 1378) Sigma Chemical Co, USA; raffinose (Pentahydrat), Merck, Germany. All other chemicals were of analytical grade. All solutions were made up with water distilled twice in a quartz apparatus.
RESULTS As is shown in table 1, three of the four studied nucleoside triphosphates induced increased frequency of association when added without activator to washed spermatozoa in the Tris-HCl buffered medium. The deviating nucleotide was CTP. In a second series of experiments of the same kind all four nucleoside triphosphates significantly increased the association. Further addition of CaZ+ caused association to increase significantly in all 4 cases. The effects of the different nucleoside triphosphates did not differ significantly from that of ATP (table 2). ADP was studied in similar experiments to those of table 1, although in phosphate buffered medium. When added without activator to the spermatozoa, ADP decreased association in one experiment and increased it in 4 experiments out of nine (table 3). In 4 experiments there was no significant change. When additions of Mg2+ or Ca2+ in final cont. of 2 x 10e6, 7 x 10~~ or 2 x 10-j M were also made, the activators significantly increased association except in the case of 2 % 1O-6 M Mg2+ (table 3). It should be observed that the final concentration of ADP in the actual experiments was 2 x 1O-4 M. The experiments of table 4 indicate that the effects of ADP shown in table 3 appeared also in the absence of phosphate at 10 times lower concentration of ADP. The same ef-
Nucleotide specificity of head-to-head association
49
Table 1. Effects of some nucleoside triphosphdtes aloize and in combination with Cazf on the frequency of association in washed bull spermatozoa in Tris-HCI-buffered
medium
Final concentrations of both nucleoside triphosphates and Ca 2+2 x 1O-5M. For the UTP-experiment 2 suspensions from one ejaculate and one suspension from another ejaculate were used; for the other experiments 2 suspensions from one ejaculate were used
Additions Sample 1 2 3 : 3 1 2 3 1 :
None CTP CTP + Ca2+ None UTP UTP + Ca2+ None GTP GTP -1Ca2+ None ITP ITP + Caz+
Mean difference 4f,-Z,iS.E. -0.08kO.26
Deviation of q-2, from zero t
P
Mean difference &,,kS.E.
t
2.30
Deviation of &-2,-W, from zero
Effect of Ca2+, &--2)-W, +S.E.
P
0.31
t
+ 1.7110.30 - 1.79kO.24
-0.4610.20
Deviation of h-3, from zero
7.46
P
5.70
~0.05
+ 1.84kO.24 7.67
i- 1.3510.28
4.82
+ 1.38i0.22
6.27
-2.31 kO.23 10.04 < 0.001 1 -0.38kO.17 -0.65kO.19
2.24 3.42
-0.05 - 1.73 kO.34
5.09
- 2.03 iO.23
8.83
co.01
feet of ADP was exerted when the medium consisted chiefly of seminal plasma (table 5). It appears from table 6 that Mg2+ was a more efficient activator than Ca2+when ADP increased the frequency of association.
DISCUSSION In the presence of Ca2+ all of the studied nucleoside triphosphates, ATP, CTP, ITP, GTP, and UTP, induce increased head-to-
Table 2. Comparison between the potencies of some nucleoside triphosphates in inducing headto-head association in washed bull spermatozoa in presence of Ca2+ The effect of ATP is used as a standard with which the effects of the other nucleotides are compared. This is done in two different experiments with different spermatozoa. Final concentrations of nucleotides and Ca2+ 2 x 1O-5 M Deviation of B from zero Sample
Additions
Mean difference, ii&S.E.
1 2 3 4
None ATP + Ca2+ CTP + Ca2+ UTP + Ca2+
O_,1-3,= -0.96kO.24 D,,-,, = -0.70+0.14
: 3 4
None ATP + Ca2+ GTP + Ca2+ ITP +Ca2+
<,I-2, = -0.8210.15
i&,,
= -0.45f0.10
Q--3) = -0.38 )0.09 D+-4, = -0.56&0.10
t
P
5.47
Mean diff. between effect of ATP and that of other NTP 4kS.E.
&-,1-3,= 0_(1+,1-4,=
-0.14kO.21 +0.13+0.18
D,,-,,-,,-a,=
-0.27t0.20
Deviation of differences from zero
t
P
0.67 0.72 1.05
4.57
@(1-2,--(1--3) = +0.07f0.10
0.70
5.6OiO.001
l&+-(1--4) = -0.12kO.16 D,~--3,--(1-~~= +0.19+0.17
0.75
1.12
Exptl Cell Res 87 (1974)
50
P. E. Lindahl
Table 3. Effect of ADP alone and in combination Iz’ith Ca’i or Mg:“+ 011 the jr.cylrcvlr:l. of association of thrice-washed bull spermatozoa in phosphate-buffered medium In all cases when ADP is the only addition the total concentration of Ca” is 3 IO-” M, and that of Mg” probably lower. One or two ejaculates were used in each experiment consisting of one or two tests. } marks components acting simultaneously
Expt no.
Addition
Added concentration (M)
ADP ADP ADP ADP ADP Mg*+ ADP ADP Ca2+ I ADP ADP Mg2+ ADP ADP Ca2+ ADP ADP Mg2+ ADP ADP Ca2+
2 A IO-” 2 _ 10-4 2; IO-” 2 /~IO-” 2 Y 1o-4 2 I: IO-6 2 >:IO-4 2 _’ IO-” 2 - 10-e 2 :
1
5 6
1 1 1 1
I
8 9
Mean difference D-tS.E.
Deviation of d from zero
Significance of effect of addition of activator
t
4kS.E.
P
1.2OkO.29 0.77 t-o.37 0.33 iO.20 -0.55+0.19 m-0.54&0.25
4.14 2.08 1.65 2.18 2.16
-0.18kO.20 -1.34-to.41
3.27 0.90
~=o.ol
em0.15+0.29 ~ 2.05 -to.49
4.18 0.52
-rO.Ol
-0.37kO.14 -- 1.00+0.22
4.55 2.64
:m0.46f0.21 1.61kO.26 - 1.13iO.25 -2.56kO.63
t
P
-.O.Ol (< - 0.02 0.05)
0.01 kO.28
0.04
- 1.16iO.41
2.83
‘. 0.02
~ 1.90+0.40
4.75
- 0.001
co.01 < 0.05 I
-0.63 kO.19
3.32
-0.01
2.19 6.19
-< 0.05 0.001
- 2.07 + 0.23
9.00
4.52 4.06
-z0.01 < 0.01
- 1.43 20.65
2.20
head association (table 1). The substrate specificity is thus rather low and independent of whether the base of the nucleoside triphos-
> 1
I
0.001 ==0.05
phate is a purine or a pyrimidine. The reason for using Ca2+ as activator, instead of Mg2+, was the fact that Ca2+ has turned out to be
Table 4. Effect of ADP alone or in combination with Ca2+ on the frequency of association of thrice-washed bull spermatozoa in Tris-HCI buffered medium One ejaculate was used in each experiment. } marks components present simultaneously
Expt no.
1 2 3
Addition
i { {
ADP ADP Ca2+ ADP ADP Ca2+ ADP ADP Ca2+
Concentration (M)
Deviation of 4 from zero Mean difference t D f S.E.
1 1 2 s10-5 1 2 X 10-S 2 x 10-b 2 x 10-S 2 x 10-S 2 x 10-h 2 x 10-E 2 x 10-S 2 x 10-s
Exptl Cell Res 87 (1974)
Significance of Ca”+-effect
P
-0.52kO.24
2.17
J 0.05
- 1.61iO.21 + 0.07 * 0.22
7.67 0.32
< 0.001
-0.68f0.15 -0.7220.22
4.53 3.27
co.01 co.01
- 1.31 kO.28
4.68
< 0.001
3 J
DkS.E.
t
P
- 1.09+ 0.24
4.54
‘- 0.01
-0.75+0.19
3.95
.-0.01
-0.59kO.17
3.47
‘--0.01
Nucleotide specificity of head- to-head association Table 5. Effect of ADP alone on the frequency of association of bull spermatozoa in 4 parts of seminal plasma and one part of phosphatebuffered medium One ejaculate was used in each experiment
Expt no. 1 2
Deviation of Concentra4 from zero tion of ADP Mean difference 4kS.E. t P CM) 2 x 10-b 2 x 10-b
-0.50+0.15 -0.33kO.09
3.33 3.65
-z0.01 < 0.01
more efficient in studies with ATP [2]. Similar properties have been demonstrated for purified actomyosin [l, 7, 16, 18, 201. When ATP is supplied as only addition to washed spermatozoa in phosphate or TrisHCl buffered medium, no association is induced [9]. As ATP does induce association when Mg2+ or Ca2+ is also added, the fact that this process failed to appear without addition of activators was looked upon as resulting from too low a concentration of the two activators in the media used. All the studied nucleoside triphosphates were able to induce association when added alone; although, for CTP this was true only in one case out of two. We thus find that ATP and,
51
although to a lesser degree, also CTP, in which compounds the 6-position in the purine and pyrimidine rings, respectively, is substituted with NH,-, need a higher concentration of Ca2+ than those in which this position is substituted with OH-. A corresponding grouping of nucleoside triphosphates appears in studies by Hasselbath [4] of contraction of actomyosin gel and simultaneous hydrolyses of the triphosphates into corresponding diphosphates and Pi. According to this author the developed tension was large, the rate of hydrolyses high, and the need for Mg2+ little with ATP and CTP, whereas with UTP, ITP, and GTP tension was small, the rate of hydrolyses high, and the need for Mg2+ great. The rate of hydrolyses of the different nucleoside triphosphates increased with increasing concentration of Mg2+ until an optimum concentration was reached. At intermediate concentrations of Mg2+ the rates of hydrolyses form a series of decreasing values from ATP to GTP in the order ATP>CTP>UTP> ITP > GTP [4]. A corresponding variation in the capacity of the different nucleoside triphosphates to induce head-to-head association at a certain concentration of Ca2+ was not observed (table 2).
Table 6. Comparison between the abilities of A4g2+ and Ca2f to induce head-to-head association in the presence of ADP Medium based on Tris-HC1 buffer. Spermatozoa from one ejaculate used in each experiment
Expt no.
Additions ADP
1
Mg2+ t ADP Ca2+ ADP
2
1 ADP Mg2+ Ca2+
Concentra- Mean tion difference ii+S.E. CM) 2x10-4 x 1 IO-5 21 x 10-s 10-a 1 x 10-S X 10-s 11 x 10-S 1 x 10-S
Deviation from zero
_
-
Deviation from zero
D~gz+-D&+
t
P
-1.18iO.22
5.36
-0.72kO.17
4.24
< 0.01
-0.9lkO.19
4.79
< 0.001
-0.42+0.11
3.82
< 0.01
*SE.
t
P
+0.46?0.17
2.71
< 0.05
+0.4810.10
4.80
< 0.001
Exptl Cell Res 87 (1974)
52
P. E. Linda111
The need for a higher concentration of CaZ+ in the cases of ATP and CTP than in the cases of ITP, GTP and UTP may depend on a similar structure of the active centre in the ATPase of the acrosome covering membrane to that suggested for myosin ATPase. The conception of the mentioned structure was based on studies of the interaction between myosin and its substrates under the influence of different reagents (modifiers; for review see Perry [ 151p. 481). Substances containing enone structure, among others the pyrimidines cytosine and uracil and the nucleoside guanosine, were found to block the “ATP-dependent” reaction [9]. This property seems to be suppressed in some way when the corresponding nucleoside triphosphates function as substrates. In a previous study [8] ADP was found to induce decrease in the frequency of association in twice washed spermatozoa. When this experiment was repeated some years later in the same manner, there was an increase in the frequency of association. It thus seemed necessary to study the effect of ADP on a broader scale and with varied concentration of Mg2+ and Ca2+ (table 3). Decreased frequency of association was effected only in one case out of 9 by ADP alone, indicating that this is a possible but rare consequence of this treatment. Obviously there is a great variability between different sperm samples, as at this low concentration of activators (cf p. 48) no significant effect appeared in 4 cases, while increased association appeared in 4 cases. In this respect ADP differs from ATP. It should, however, be stressed that the concentration of ADP in the present experiments was 10 times higher than that in the corresponding previous experiments with ATP [lo]. With one exception, addition of low concentrations of Mg2+ and Ca2+ to samples containing ADP induced further increase in Exptl Cell Res 87 (1974)
association (table 3). In order to rule out the possibility that the relatively high concentration of ADP and the buffer system used, cooperated in or contributed to the increased association appearing in table 3, a few similar experiments were performed with a 10 times lower concentration of ADP and the Tris-HC1 buffered medium (table 4). The results were rather similar to those in table 3, indicating that neither the higher concentration of ADP nor of the phosphate buffer are of any importance for the observed effect of ADP. The original experiments with ADP, referred to [8], were performed with spermatozoa washed only twice. The concentrations of seminal plasma components might have been higher in those experiments than in the present ones. Therefore the effect of ADP as only addition to seminal plasma, diluted 4 parts to one part of phosphate-buffered medium (table 5) was studied. It should be pointed out that seminal plasma contains Mg2+ and Ca2+ in concentrations high enough to permit these ions to function as activators of the ATP-dependent reaction (cf [IO] p. 428). The effect of ADP was just the same in the presence of seminal plasma components (table 5) as in their absence (table 3) and these do not seem to have any special influence. The comparatively higher effect of Mg’-1 than of Ca2+ in pairs 6-7 and 8-9 of experiments referred to in table 3 suggests that Mg2+ is more efficient than Ca2+ in causing head-to-head association in the presence of ADP. Experiments in which both ions were tested with spermatozoa from the same suspension confirm this suggestion (table 6). As the hypothesis upon which the present investigations originally were based implies that head-to-head association is connected with hydrolyses of ATP, the present results should be discussed against this background. Some different possibilities concerning the
Nucleotide specificity of head-to-head association enzymic splitting of ATP, may be discussed. (1) ADP is hydrolyzed by the same enzyme as ATP. If this is the case it is remarkable that Mg2+ is a more efficient activator than Ca2+, whereas this is reversed when ATP functions as the substrate. These facts might possibly depend on the relevant stability constants for the two nucleotides and the cations in question. However, inspection of the constants [19] reveal relationships suggesting that the most efficient activator should be Mg2+ for ATP and Ca2+ for ADP, i.e., just the opposite to what was found in the experiments. (2) ADP is transformed into ATP and AMP by adenylate kinase. If this reaction is slower than the splitting of ATP, it will determine the rate of hydrolyses of ATP when ADP is the only exogenous substrate. As a matter of fact, Mg2+ is more efficient than Ca2+ as activator of adenylate kinase from most tissues, although the reverse has also been observed [13]. This manner of reasoning implies that the adenylate kinase should be on the outside of the acrosome covering membrane (cf below). (3) ADP acts in quite a different way, e.g. by increasing the permeability for ATP in the mentioned membrane, thus releasing ATP (and activators) from the interior of the cell. Because of the relatively fast action of exogenous ATP on head-to-head association it was inferred that this slowly penetrating substance must act on the outer surface of the cell membrane [lo]. This conclusion should be extended to other nucleoside triphosphates and most probably also to ADP. Thus, when scrutinizing the literature for enzymes hydrolyzing nucleoside triphosphates and ADP, only statements referring to intact spermatozoa are of interest for the current problem. Further, we confine ourselves to
53
information concerning Mg2+- or/and Ca2+activated ATPase, as further addition of Na++K+ had no effect on head-to-head association that could not be related to increased ionic strength [lo]. The above-mentioned conditions are fulfilled by experiments with spermatozoa from three species, viz., bull [14, 171, ram [17, 211 and man [3]. In all cases the ATPase was activated by Mg2+. Ca2+ was also effective in bull [3] and ram [21] spermatozoa, in the latter study more so than Mg2+. ADP was hydrolysed by bull [14], ram [21] and human [14] spermatozoa. According to Vogelmayr et al. [21] negligible amounts of AMP were found when both ATP and ADP were present at the same time, “indicating a preferential hydrolysis of ATP”. With human spermatozoa [3] 80% of the added ATP was hydrolysed (37°C) after 20 min and about twice as much ADP as AMP was present. CTP and ITP were hydrolysed by bull spermatozoa [14] and, by human spermatozoa, AMP too was dephosphorylated
131. Observations made with spermatozoa of one species may not be generalized to apply to all three species, and can only be stated tentatively. If the acrosome covering membrane has the same enzymic outfit as the rest of the surface membrane then the interpretation of the present observations on head-tohead association, as resulting from enzymic splitting of certain substrates, is well in keeping with the facts reported above from other investigations. Nelson [12] proposed the occurrence of adenylate kinase in bull spermatozoa, and this would provide an easy explanation for the results [3, 14, 211 accounted for above (cf [14]). To my knowledge, this enzyme has still not been definitely demonstrated in mammal spermatozoa. It is nevertheless of interest to note that the dephosphorylation Exptl Cell Res 87 (1974)
54
P. E. Linduhl
rates, when ADP
is the only substrate, are 209); lower than with ATP alone [21]. Such a relationship was assumed as an explanation of the fact that Mg”+mwas a more efficient activator than Ca2+ when the association was induced by ADP (p. 53). The great variation in the results obtained with ADP as only addition to washed spermatozoa may possibly have some relationship to the degree of maturation or senescence of the spermatozoa in different ejaculates. This would mean that these processes lead to changes in the permeability of divalent cations in the part of the cell membrane in question. 1 am indebted to Dr T. Svensson, former Chief Veterinarian of the AI-Station at Enkoping, and to Dr B. Gustafsson. Head of the Clinic for Obstetrics and Gynaecology,‘the Veterinary College, Stockholm, for generous supply of bull semen. I thank Mrs Gun Ronnqvist for very efficient assistance. This investigation was supported by the Swedish Natural Science Research Council.
Bettex-Galland, M & Ltisher, E F. Advances in protein them 20 (1965) 1. 3. Durr, I F, Abla, A & Mroueh, A. J reprod fert 31 (1972) 313. 4. Hasselbach. W. Biochim bioohvs - acta 20 (1956) 355. 5. Jones, B M. Nature 212 (1966) 362. 6. Jones, P C T, Nature 212 (1966) 365. 7. Kiellev, W W & Blum. J J. Arch biochem biophys 55 (1955) 486. 8. Lindahl, P E, Exptl cell res 53 (I 968) 626. 9. -- Ibid 53 (1968) 639. 10. - Ibid 81 (1973) 413. II. Lindahl, P E, Bodin, N 0 & Brattsand, R, lnt j fert 8 (1963) 825. 12. Nelson, L, J cell physiol 68 (1966) 113. 13. Noda, L, The enzymes (ed P D Boyer) vol. 8, p. 279. Academic Press, New York (1973). 14. O’Donnell, J M & Ellory, J X, Biochem sot (London), agenda papers 8-9 June 1972, p. 17. 15. Perry, S V, Ann rev biochem 30 (1961) 473. 16. Portzehl, H, Biochim biophys acta 14 (1954) 195. 17. Quinn, P J & White, I G, J reprod fert 15 (1968) 449. 18. Ranney, R E, Am j physiol 178 (1954) 517. 19. Sillen, L G & Martell, A (compilers), Stability constants of metal ion complexes. 2nd edn. Special pub1 No 17. The Chemical Society, London (1964). 20. Spicer, S S & Bowen, W J, J biol them 188 (1951) 741. 21. Vogelmayr, J K, Quinn, P J & White, I G, Biol reprod 1 (1969) 215. 2.
REFERENCES 1. Bergkvist, R & Deutsch, A, Acta them Stand 8 (1954) 1105.
Exptl Cell Res 87 (1974)
Received February 1, 1974