- Email: [email protected]
Heavy metals and spermatozoan motility
Experimental Cell Research83 (1974) 87-94
HEAVY METALS AND SPERMATOZOAN
MOTILITY
II. Turbidity Changes Induced by Divalent Cations and
Adenosinetriphosphate in Sea Urchin Sperm Flagella M. MORISAWAl Department
of Biology,
University
and H. MOHRI
of Tok.vo, Komaha, Megwo-ku,
Tokyo, Japan
SUMMARY A marked increase in turbidity was caused when heavy metals, such as Cd*+ and Zn”-, in relatively low concentrations were added to suspensions of either intact flagella or axonemes isolated from sea urchin spermatozoa. Mg2+ and Cazc were much less effective, while Na+ and K’ were without effect. The turbidity increase was almost abolished by adding equivalent amounts of EDTA or pvrophosphate. Addition of ATP to the metal-treated suspensions of flagella also brought about a decrease in turbidity. ADP was not as effective as ATP and little decrease was observed with AMP. The same was true with other nucleoside phosphates. Within a certain range of concentrations, Cd2+ and Zn2+ caused fluctuations in the turbidity, which gradually decayed. ATP restored the fluctuations which had once disappeared. The phenomenon could not be observed with the samples after long standing, heating, sonication or treatment with 8 M urea. Similar results were obtained with suspensions of microtubules isolated from the flagella. The observed changes in turbidity are discussed in connection with flagellar movement.
Almost twenty years ago, Fujii and his school reported the presence of zinc in both spermatozoa and dividing cells such as sea urchin eggs and stamina1 hair cells of Tradescantiu, and postulated that the metal would play some important role in these cells [2-51. Since then, however, the problem has long been left unresolved. At present it is well known that sperm tails (flagella) and the mitotic apparatus of dividing cells have in common a major component consisting of microtubules, which seem to be one of the essential elements for cell motility. We have tried, therefore, to find some relationship between zinc and flagella or microtubules. 1 Present address: Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo, Japan.
In fact, it was found that the flagella and their microtubules isolated from sea urchin spermatozoa contain zinc in relatively high concentrations [ 141. In sea urchin eggs, on the other hand, Sakai [I 51 reported that a thread model prepared by introducing a KCLsoluble protein fraction of the eggs into acetone or distilled water repeatedly contracted and relaxed on the alternate addition of metal ions, including zinc, and chelating agents. This was interpreted as due to electrostatic forces between negative charges of the protein and added metal ions. However, attempts to obtain such a contractile gel thread from flagella of sea urchin spermatozoa have not so far been successful, nor can such a thread be prepared from the material derived from isolated Exptl Cell Res 83 (1974)
88
M. Morisawa & H. Mohri
mitotic apparatus of sea urchin eggs [16]. We have therefore looked for another system which could provide information concerning the effects of metals on the sperm flagella. The structural changes, swelling and contraction, of mitochondria have on occasion been followed by measuring the optical density of their suspensions. In the isolated flagella from fish spermatozoa, Tibbs [21] observed a reduction in turbidity of the flagellar suspensions caused by ATP, which was explained as due to a swelling of the flagella. This observation was further substantiated by weighing the centrifugal pellets of the flagellar suspensions after treatment with ATP [22-241. A similar result has been reported with respect to cilia and their fractions isolated from Tetrahymena [7]. Electron microscopical observations revealed that ATP caused disintegration of the flagella into groups of microtubules or individual microtubules [20, 241. In the present work, we have tried to examine the effects of metals and chelating agents, especially ATP, on flagella and microtubules by following the changes in turbidity. Within a certain range of concentrations, zinc and ATP were found to cause an oscillation in turbidity of the flagellar suspensions. These results are considered in relation to spermatozoan motility. MATERIALS
AND METHODS
Spermatozoa of the sea urchins, Anthocidaris rrassispina, Pseadocentrotus depresuss and Hemicentrotus pulcherrimus, were used as materials. Flagella were isolated from mechanically disrupted spermatozoa as described previously [9, 121. Axonemes (flagella deprived of membranes) and microtubules were prepared according to the method of Gibbons [6] with some modifications. Namely, the isolated flagella were extracted with either 0.5 sb digitonin or- 1 “4, Triton X-100 in 30 mM Tris-HCI buffer, pH 8.2, to remove flagellar membranes, washed with the buffer solution and then dialysed against a mixture of I mM Tris-HCI, pH 8.2, 0.1 mM EDTA and 1 mM p-mercaptoethanol, which leaves the outer doublet microtubules undissolved. Flagella, axonemes Exptl Cell Res 83 (1974)
or microtubules were finally suspended in I mM Tris-HCI, pH 8.2. Rabbit skeletal muscle actin was the kind gift from Professor K. Maruyama, Kyoto University. Actin was also prepared from the acetone powder of thoraces of flesh-fly, Boettcherisca peregrina. Turbidity changes were followed by measuring either quasi-attenuance in a Hitachi Perkin-Elmer spectrophotometer, or rectilinear attenuance in a Shimazu Multi-purpose spectrophotometer, at 370 nm, using I cm cuvettes at room temperature. No essential difference was observed between the results obtained with these two methods, except that somewhat higher values were recorded with the latter for the same suspension. The suspensions for turbidity measurements were prepared in 20 mM Tris-HCI buffer, pH 7.5 or 8.2: In some experiments Tris-HCI buffer was replaced by HEPES (N-2-hvdroxvethvlpiperazine-N’-ethanesujfonic acid)-NaOH buffer, pH 7.5, which does not chelate metals. Protein contents were measured by the method of Lowry et al. [8]. Homogenates were prepared in a Teflon homogenizer. Sonication was done with a 20 KC Branson sonifier. All chemicals were commerciallv available products. Nucleoside phosphates were purchased from Boehringer Mannheim Japan, K. K. The metals used were in the form of chlorides. Distilled deionized water (DDW) was used to prepare solutions.
RESULTS Effects of metal ions The effects on the turbidity of suspensions of intact flagella, axonemes and microtubules were examined with several monovalent and divalent cations, including Na+. K+, Mg2+, Ca2-, Sr”+, Mn2+, Fez+, Co2+, Ni”+, Cu”“. Zn2+, Cd2+ and Hg’+. As can be seen from fig. 1, the turbidity of these suspensions was increased by addition of divalent cations at the concentration of 0.5 mM, which was effective in inducing contraction of gel threads prepared from sea urchin eggs [15]. In the case of Fez+, the optical density due to the metal itself was subtracted. In all cases, the initial rapid increase in the turbidity gradually subsided, reaching a plateau within about 10 min. It appeared that there was no significant difference among the suspensions of intact flagella, axonemes and microtubules. When 20 mM Tris-HCl buffer, pH 7.5 or 8.2, was used as the suspending medium,
Heavy metals and sperm motility.
the efficiency of divalent metal ions in causing an increase in turbidity of the suspensions showed the following order, if added at the Cd2+ > Cu2+ > Zn”+ > same concentration: Fe’+ > Hg2+ > Ni2+ > Co2+ > Mn2+ > Sr2+ > Ca”+ > Mg’+, although the order was sometimes reversed between two adjacent metals, e.g. between Ni2+ and Co2+. The monovalent cations Na+ and Kf, on the other hand. had little effect on the turbidity even when their concentrations were raised to 0.1 M or more. When Tris-HCI buffer was replaced by HEPES buffer, pH 7.5, the turbidity increase was more pronounced at the same concentration of these metals, suggesting some chelating action of Tris buffer. Ejyects of EDTA, nucleotides
pyrophosphate
2
4
6
8
-~~-~~~
8
12
10
Fig. 2. Abscissa: time (min); ordinute: OD.
Effects of pyrophosphate, EDTA and adenosine or guanosine nucleotides in reducing the turbidity increase of axoneme suspensions caused by zinc or cadmium (Anthocidaris). 20 mM Tris-HCI buffer, pH 8.2; 0.44 mg protein/3 ml; 0.5 mM ZnCl, or CdCI,; 0.66 mM pyrophosphate, EDTA and nucleotides. I, AMP; 2, ADP; 3, ATP; 4, GMP; 5, GDP; 6, GTP; 7, pyrophosphate; 8, EDTA.
and
The increase in turbidity caused by the metal ions was reversed by the addition of chelating agents, such as EDTA, pyrophosphate and also nucleoside phosphates (figs 1, 2). The reduction in turbidity was observed when the concentration of these agents exceeded that of the metals previously added, indicating a one-to-one relationship between the metals
0
5
89
II
10
12
Fig. 1. Abscissa: time (min); ordinare: OD. Effects of monovalent or divalent cations and ATP on the turbidity of axoneme suspensions (Anrhocidavis). 20 mM Tris-HCI buffer, pH 7.5; 0.44 mg protein/3 ml; 0.5 mM monovalent or divalent cations; 0.66 mM ATP.
and the chelating agents. It was found that the depressing effect of these reagents when Zn2+ or Cd2+, for instance, were used as the cations, diminished in the following order: phosEDTA > pyrophosphate > nucleoside phates. When Hg2+ was added, however, the once-elevated turbidity of the suspensions was only slightly reduced by adding ATP. This might be due, at least in part, to the strong affinity between this metal and the flagellar proteins. A similar, although less marked. result was obtained in the case of Fez+. As shown in fig. 2, ADP was also effective in reducing the turbidity increase due to either Zn2+ or Cd2+, but its effect was about half that of ATP. AMP was almost without effect. The same was true of the guanosine phosphates GTP, GDP and GMP. GTP as well as ITP and CTP decreased the turbidity to almost the same extent as ATP. The effect of UTP, however, was less than those of the other nucleoside triphosphates. Thus, there was found a parallelism between the depressing effect and the strength of chelating action. Exptl Cell Res 83 (1974)
90
M. Morisawa & H. Mohri
Fig. 3. Abscissa: time (mm); ordinate:
OD. Fluctuations in turbidity of axoneme suspensions induced by zinc or cadmium (Pseudocenfrotus). 20 mM Tris-HCI buffer, pH 8.2; 0.44 mg protein/3 ml; 0.05 mM ZnC1, or CdCI, were added at time 0.
Fluctuations in turbidity When the effect of several concentrations of Cd*+ on the turbidity of the microtubule suspensions was examined, it was found that at Cd2+ concentrations around 0.05 mM the turbidity alternately increased and decreased with a periodicity of a few minutes. The fluctuation gradually subsided and the turbidity finally reached a certain level dependent on the amount of metal added. As shown in fig. 3, the same was true with the suspensions of axonemes. Zinc as well as cadmium caused fluctuations in turbidity. Under the experimental conditions examined, the periodical change appeared most markedly at protein concentrations of about 0.15-0.2 mg/ml, suggesting a stoichiometric relationship between the added metal and the flagellar protein in producing the observed changes in turbidity. In these experiments, cadmium was used because it belongs to the same column as zinc on the periodic table, and was the most effective in increasing the turbidity of the suspensions. As described above, mercury, another metal in the same column, combined Exptl Cell Res 83 (1974)
irreversibly with the flagellar protein. Of these three metals, only zinc was found in flagella in considerable amounts [14]. Iron, which was also found in flagella, was not effective in causing the fluctuation in turbidity If ATP was added to the suspensions of axonemes (fig. 4) the turbidity of which had become stable after the initial addition of Zn2+. the fluctuations of turbidity reappeared. In this case, also, a one-to-one relationship was found between Zn2+ and ATP, i.e.. 0.05 mM ATP was the most effective in the presence of 0.05 mM ZnCI,. The use of aged axonemes in this particular experiment resulted in the disappearance of the typical fluctuation pattern when Zn’+ alone was present (see below). Restoration of the fluctuations was also caused by GTP and ADP, but to a much lesser extent than that caused by ATP. AMP was not effective. In all these cases, agitation of the suspensions with a glass rod during the measurements of turbidity failed to alter the fluctuation patterns, nor could any heavy precipitates be seen in the suspensions. The fluctuations, therefore, cannot be ascribed to convection currents in the cuvettes.
Ln
ATP
5 I
Fig. 4. Abscissn: time (min); ordinate: OD. Restoration by ATP of the fluctuations in turbidity of axoneme suspensions (Anthocidoris). 20 mM, Tris-HCI buffer, pH 8.2; 0.44 mg protein/3 ml; 0.05 mM &Cl,; ATP cont.: I, 0.05 mM; 2, 0.10 mM; 3, 0.20 mM; 4, 0.30 mM; 5, 0.50 mM.
Heavy metals and sperm motility. II
‘ATP
(0)
35
3oo: I 0
.34
2
4
6
8
IO
12
14
16
‘ATP 11j
(b)
I 2
30
-2 0
2
4
6
8
IO
12
14
16
Fig. 5. Abscissu: time (min); ordinate: OD. Effects of incubation at various temperatures on the fluctuation patterns of axoneme suspensions (Anthociduris). 20 mM Tris-HCI buffer, pH 8.2; 0.44 mg protein13 ml; 0.05 mM ZnCl,; 0.05 mM ATP. to) incubation at 0°C; I, for 12; 2, 22; 3, 36 h; (b) heat treatment. I, 37 C, 60 min; 2, 0 C, 0 min; 3, 57 C 30 min; 4, IOO’C, 60 min.
Factors influencing fluctuations in turhiditj In the following experiments, the effects of various factors were examined on the fluctuation which reappeared after the addition of 0.05 mM ATP, as such a fluctuation could be observed more consistently than the initial fluctuation in the presence of 0.05 mM Zn”+ or Cd2+ alone. As shown in fig. 5a, when the axoneme suspensions were stored at 0°C for varying lengths of time, the amplitude and frequency of the fluctuation became smaller in proportion to the length of time. The same was true when the temperature for standing the suspensions was increased. It was further ernphasized from the turbidity pattern in heat-treated axoneme suspensions (fig. 5b) that thermal denaturation of the flagellar proteins results in decrease or disappearance of the fluctuations.
91
When the axoneme suspensions were sonicated, the optical densities before and after the addition of Zn2+ decreased progressively with increasing time of sonication, which disintegrates the flagellar structures. Furthermore, the intensity of the fluctuations in the presence of ATP was gradually diminished and no fluctuations were observed after 2 min sonication. A solution of axonemes dissolved in 8 M urea did not show any fluctuations in turbidity. When the dissolved axonemes were dialysed against DDW for 30 h to remove the urea, however, the fluctuation pattern could be observed, although the amplitude of fluctuation was smaller than that of the intact axoneme suspension (fig. 6). The difference between these two cases suggests that the flagellar structures, once disrupted by 8 M urea, might have been reconstituted to some extent during the course of dialysis (see also
VW. Bovine serum albumin, which was examined instead of flagellar axonemes and microtubules, failed to show the fluctuations in turbidity. The results of subjecting Gactin and F-actin from rabbit skeletal muscle and insect flight muscle to the conditions
03
.oor--.
L-
O
3
2
4
6
8
10
12
14
16
Fig. 6. Abscissa: time (mm); ordinate: OD. Effects of urea treatment on the fluctuation patterns of axoneme suspensions (Anthocidoris). The experimental conditions were the same as in fig. 5. The figure shows the fluctuation oatterns observed after the addition of ATP. I, control, dialysed against DDW for 30 h; 2, dissolved in 8 M urea and then dialysed against DDW for 30 h; 3, dissolved in 8 M
urea.
Exptl Cell Res 83 (1974)
92
M. Morisawa & H. Mohri
under which fluctuations in turbidity were observed with axonemes or microtubules were also negative. DISCUSSION From the results obtained in the foregoing experiments, the change in turbidity induced by divalent cations and chelating agents seems to be analogous to the shortening and elongation of the thread model prepared from sea urchin eggs. In fact, the order of efficiencies of the divalent cations in causing contraction of the thread mode1 is similar to the order in which they increase the turbidity of flagellar suspensions [ 151. Furthermore, ATP failed to induce contraction of the thread mode1 in the presence of K+ and Mg2+. Instead, the nucleotide caused elongation of the mode1 which had shortened on the addition of divalent cations [26]. It has been pointed out, in connection with the thread model, that some metal ions other than Cu2+ and Fe’+, such as Cd2+, Zn2+ and Hg2+, would be expected to combine directly with -SH groups, forming mercaptide [15]. Such a mechanism might well be responsible for the changes in turbidity of the flagellar suspensions in addition to electrostatic attraction, affecting intermicrotubular, intermolecular or intramolecular connections. Our previous paper [14] reported that zinc and iron are concentrated in sea urchin sperm flagella. However, iron was found to combine rather irreversibly with the flagellar components and did not induce the fluctuations in turbidity. The observation that zinc combines with the flagellar protein in some fashion that is reversible by ATP suggests that this metal plays role in spermatozoan motility. Further differences between zinc and iron in their effects on the conformation of flagellar proteins will be shown in a following paper [I 31. Exptl
Ceil Res 83 (1974)
The intial fluctuations induced by minute amounts of Zn2+ or Cd2+ would arise from interactions between the added metal ions and the nucleotides contained in these preparations. The subsequent gradual subsidence of the fluctuations would then be associated with the decrease in the amount of such nucleotides, which could affect the binding of the metal ions to the flagellar proteins. In fact, it has been reported that guanosine nucleotides are present in isolated flagella and microtubules [ 11, 19,251, while adenosine nucleotides are apt to be washed out during the course of preparation. Alternatively. the synchronism of some phenomenon underlying the fluctuations in turbidity, once established by adding the metal ions, might be gradually disturbed. The restoration of the periodical changes in turbidity by the addition of equivalent amounts of ATP indicates that both the metal ions and the nucleotides are indispensable for the oscillation. It is quite probable that the periodical fluctuations are ‘physiological’, because they gradually disappeared with the lapse of time at various temperatures and on treatment with heat, ultrasonic waves or urea; moreover, they seem to be specific for the flagellar components. The phenomenon would be an in vitro expression of the rhythmic wave motion of sperm flagella, although the frequency of the fluctuations observed was much less than that of actual flagellar movement. This could be interpreted as due to loosening of the tight coupling between the flagellar components or loss of some factor(s) essential for flagellar movement. Tn recent years, considerable information has been accumulated concerning the mode of movement of flagella and cilia on the one hand, and the properties of their components, flagellar ATPase (dynein) and microtubule protein (tubulin), on the other. The predominant hypothesis presented so far to account
Heavy metals and sperm motility. II for the basic mechanisms of flagellar movement is the sliding theory, which was originally applied to muscle contraction [17], although there is also another hypothesis, which assumes the occurrence of contraction and relaxation of flagellar microtubules or their active units [18, 251. More recently, Summers & Gibbons [20] have shown that active sliding occurs between groups of the outer doublet microtubules of trypsin-treated sea urchin sperm flagella in the presence of ATP and magnesium. In any case, it is unquestionable that the flagellar microtubules are bent with a certain curvature, either passively or actively. Since analyses of the wave motion of sperm flagella have indicated that it consists of circular arcs combined with straight lines [I], a certain structural unit (tubulin molecule?) on a fixed point of flagella or microtubules would be expected to oscillate between states corresponding to ‘bend’ and ‘unbend’. The bending would be associated with changes in the interactions among flagellar structures and/or some conformational changes of the flagellar proteins. It can be assumed that the continuous combination and removal, by ATP, of zinc ions at certain binding sites on the flagellar proteins would cause cyclic changes in their conformation, resulting in the fluctuations in turbidity as an expression of such periodical changes at the macroscopic level. That the oscillation in the turbidity could be observed with a fraction containing only microtubules implies that the phenomenon takes place in the absence of dynein ATPase and is probably due to an intrinsic property of the microtubule protein. Since, however, the chelating activity of ADP is much less than that of ATP, the action of dynein ATPase would allow the zinc ions once released by ATP to recombine with the microtubule proteins, thus exaggerating the periodical changes.
93
In the present experiment, electron microscopical observations appeared to suggest that the periodical increase and decrease in turbidity might be due to aggregation and disaggregation of the flagellar microtubules. It is quite possible, however, that the changes in turbidity induced by heavy metals and chelating agents, especially zinc and ATP, would reflect not only the mutual aggregation or disaggregation of flagellar components, but also conformational changes in the flagellar proteins. Some promising results which have been obtained in this connection will be reported in a forthcoming paper
u31. The authors wish to thank Dr J. C. Dan for her kind help in preparing the manuscript. They are also indebted to Miss M. Shimomura for her technical assistance.Finally, the authors expresstheir gratitude to ProfessorH. Sugawarafor his kind interest and warm encouragement. This work was supported by grants-in-aid from the Ministry of Education, Japan, and grants (No. M67.0140, No. M69.0130, No. M71.08C, No. M72.051C)from the Population Council, Inc., USA. REFERENCES 1. Brokaw, C J, J exptl biol 43 (1965) 155. 7 Fujii, T, Annot zool japon 27 (1954) 115. 5: - J fat sci univ Tokyo IV 7 (1953) 224. 4. - Ibid IV 7 (1955)327. 5. Fujii, T, Utida, S, Mizuno, T & Nanao, S, J fat sci univ Tokyo IV 7 (1955)335. 6. Gibbons, I R, Arch biol (Littge) 76 (1965)317. 7. - J cell biol 26 (1965)707. 8. Lowry. 0 H, Rosebrough, N J, Farr, A L & Randall, R J,‘J biol them 193 (1951) 265. 9. Mohri, H, Biol bull 127(1964)381. IO. Mohri, H, Murakami, ‘s &’ Maruyama, K, J biochem 61 (1967)518. I I. Mohri, H & Yanagisawa,T, Zoo1mag 76 (1967)
276. 12. Morisawa, M, Zoo1mag 78 (I 969)484. 13. - In ureoaration. 14. Morisawa, M & Mohri, H, Exptl cell res 70 (1972) 311. 15. Sakai, H, J gen physiol 45 (1962)412. 16. - Personalcommunication. 17. Satir, P, J cell biol 39 (1968) 77. 18. Silvester, N R & Holwill, M E J, Nature 205 (I 965) 665. 19. Stephens, R E, Renaud, F L & Gibbons, I R,
Science156(1967)1606.
20. Summers, K E & Gibbons, I R, Proc natl acad sci US 68 (1971) 3092. Exptl Cell Res 83 (1974)
94
M. Morisawa
& H. Mohri
21. Tibbs, J, Spermatozoan motility (ed D W Bishop) p. 233. Am Ass Adv Sci, Washington, D.C. (1962). 22. - Nature 193 (1962) 686. 23. - Biochem j 89 (1963) 97. 24. - Ibid 96 (1965) 340.
Exptl Cell Res 83 (1974)
25. Yanagisawa, T, Hasegawa, S & Mohri, H, Exptl cell res 52 (1968) 86. 26. Yazaki, I. Personal communication. Received July 10, 1973
HEAVY METALS AND SPERMATOZOAN
MOTILITY
II. Turbidity Changes Induced by Divalent Cations and
Adenosinetriphosphate in Sea Urchin Sperm Flagella M. MORISAWAl Department
of Biology,
University
and H. MOHRI
of Tok.vo, Komaha, Megwo-ku,
Tokyo, Japan
SUMMARY A marked increase in turbidity was caused when heavy metals, such as Cd*+ and Zn”-, in relatively low concentrations were added to suspensions of either intact flagella or axonemes isolated from sea urchin spermatozoa. Mg2+ and Cazc were much less effective, while Na+ and K’ were without effect. The turbidity increase was almost abolished by adding equivalent amounts of EDTA or pvrophosphate. Addition of ATP to the metal-treated suspensions of flagella also brought about a decrease in turbidity. ADP was not as effective as ATP and little decrease was observed with AMP. The same was true with other nucleoside phosphates. Within a certain range of concentrations, Cd2+ and Zn2+ caused fluctuations in the turbidity, which gradually decayed. ATP restored the fluctuations which had once disappeared. The phenomenon could not be observed with the samples after long standing, heating, sonication or treatment with 8 M urea. Similar results were obtained with suspensions of microtubules isolated from the flagella. The observed changes in turbidity are discussed in connection with flagellar movement.
Almost twenty years ago, Fujii and his school reported the presence of zinc in both spermatozoa and dividing cells such as sea urchin eggs and stamina1 hair cells of Tradescantiu, and postulated that the metal would play some important role in these cells [2-51. Since then, however, the problem has long been left unresolved. At present it is well known that sperm tails (flagella) and the mitotic apparatus of dividing cells have in common a major component consisting of microtubules, which seem to be one of the essential elements for cell motility. We have tried, therefore, to find some relationship between zinc and flagella or microtubules. 1 Present address: Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Nakano-ku, Tokyo, Japan.
In fact, it was found that the flagella and their microtubules isolated from sea urchin spermatozoa contain zinc in relatively high concentrations [ 141. In sea urchin eggs, on the other hand, Sakai [I 51 reported that a thread model prepared by introducing a KCLsoluble protein fraction of the eggs into acetone or distilled water repeatedly contracted and relaxed on the alternate addition of metal ions, including zinc, and chelating agents. This was interpreted as due to electrostatic forces between negative charges of the protein and added metal ions. However, attempts to obtain such a contractile gel thread from flagella of sea urchin spermatozoa have not so far been successful, nor can such a thread be prepared from the material derived from isolated Exptl Cell Res 83 (1974)
88
M. Morisawa & H. Mohri
mitotic apparatus of sea urchin eggs [16]. We have therefore looked for another system which could provide information concerning the effects of metals on the sperm flagella. The structural changes, swelling and contraction, of mitochondria have on occasion been followed by measuring the optical density of their suspensions. In the isolated flagella from fish spermatozoa, Tibbs [21] observed a reduction in turbidity of the flagellar suspensions caused by ATP, which was explained as due to a swelling of the flagella. This observation was further substantiated by weighing the centrifugal pellets of the flagellar suspensions after treatment with ATP [22-241. A similar result has been reported with respect to cilia and their fractions isolated from Tetrahymena [7]. Electron microscopical observations revealed that ATP caused disintegration of the flagella into groups of microtubules or individual microtubules [20, 241. In the present work, we have tried to examine the effects of metals and chelating agents, especially ATP, on flagella and microtubules by following the changes in turbidity. Within a certain range of concentrations, zinc and ATP were found to cause an oscillation in turbidity of the flagellar suspensions. These results are considered in relation to spermatozoan motility. MATERIALS
AND METHODS
Spermatozoa of the sea urchins, Anthocidaris rrassispina, Pseadocentrotus depresuss and Hemicentrotus pulcherrimus, were used as materials. Flagella were isolated from mechanically disrupted spermatozoa as described previously [9, 121. Axonemes (flagella deprived of membranes) and microtubules were prepared according to the method of Gibbons [6] with some modifications. Namely, the isolated flagella were extracted with either 0.5 sb digitonin or- 1 “4, Triton X-100 in 30 mM Tris-HCI buffer, pH 8.2, to remove flagellar membranes, washed with the buffer solution and then dialysed against a mixture of I mM Tris-HCI, pH 8.2, 0.1 mM EDTA and 1 mM p-mercaptoethanol, which leaves the outer doublet microtubules undissolved. Flagella, axonemes Exptl Cell Res 83 (1974)
or microtubules were finally suspended in I mM Tris-HCI, pH 8.2. Rabbit skeletal muscle actin was the kind gift from Professor K. Maruyama, Kyoto University. Actin was also prepared from the acetone powder of thoraces of flesh-fly, Boettcherisca peregrina. Turbidity changes were followed by measuring either quasi-attenuance in a Hitachi Perkin-Elmer spectrophotometer, or rectilinear attenuance in a Shimazu Multi-purpose spectrophotometer, at 370 nm, using I cm cuvettes at room temperature. No essential difference was observed between the results obtained with these two methods, except that somewhat higher values were recorded with the latter for the same suspension. The suspensions for turbidity measurements were prepared in 20 mM Tris-HCI buffer, pH 7.5 or 8.2: In some experiments Tris-HCI buffer was replaced by HEPES (N-2-hvdroxvethvlpiperazine-N’-ethanesujfonic acid)-NaOH buffer, pH 7.5, which does not chelate metals. Protein contents were measured by the method of Lowry et al. [8]. Homogenates were prepared in a Teflon homogenizer. Sonication was done with a 20 KC Branson sonifier. All chemicals were commerciallv available products. Nucleoside phosphates were purchased from Boehringer Mannheim Japan, K. K. The metals used were in the form of chlorides. Distilled deionized water (DDW) was used to prepare solutions.
RESULTS Effects of metal ions The effects on the turbidity of suspensions of intact flagella, axonemes and microtubules were examined with several monovalent and divalent cations, including Na+. K+, Mg2+, Ca2-, Sr”+, Mn2+, Fez+, Co2+, Ni”+, Cu”“. Zn2+, Cd2+ and Hg’+. As can be seen from fig. 1, the turbidity of these suspensions was increased by addition of divalent cations at the concentration of 0.5 mM, which was effective in inducing contraction of gel threads prepared from sea urchin eggs [15]. In the case of Fez+, the optical density due to the metal itself was subtracted. In all cases, the initial rapid increase in the turbidity gradually subsided, reaching a plateau within about 10 min. It appeared that there was no significant difference among the suspensions of intact flagella, axonemes and microtubules. When 20 mM Tris-HCl buffer, pH 7.5 or 8.2, was used as the suspending medium,
Heavy metals and sperm motility.
the efficiency of divalent metal ions in causing an increase in turbidity of the suspensions showed the following order, if added at the Cd2+ > Cu2+ > Zn”+ > same concentration: Fe’+ > Hg2+ > Ni2+ > Co2+ > Mn2+ > Sr2+ > Ca”+ > Mg’+, although the order was sometimes reversed between two adjacent metals, e.g. between Ni2+ and Co2+. The monovalent cations Na+ and Kf, on the other hand. had little effect on the turbidity even when their concentrations were raised to 0.1 M or more. When Tris-HCI buffer was replaced by HEPES buffer, pH 7.5, the turbidity increase was more pronounced at the same concentration of these metals, suggesting some chelating action of Tris buffer. Ejyects of EDTA, nucleotides
pyrophosphate
2
4
6
8
-~~-~~~
8
12
10
Fig. 2. Abscissa: time (min); ordinute: OD.
Effects of pyrophosphate, EDTA and adenosine or guanosine nucleotides in reducing the turbidity increase of axoneme suspensions caused by zinc or cadmium (Anthocidaris). 20 mM Tris-HCI buffer, pH 8.2; 0.44 mg protein/3 ml; 0.5 mM ZnCl, or CdCI,; 0.66 mM pyrophosphate, EDTA and nucleotides. I, AMP; 2, ADP; 3, ATP; 4, GMP; 5, GDP; 6, GTP; 7, pyrophosphate; 8, EDTA.
and
The increase in turbidity caused by the metal ions was reversed by the addition of chelating agents, such as EDTA, pyrophosphate and also nucleoside phosphates (figs 1, 2). The reduction in turbidity was observed when the concentration of these agents exceeded that of the metals previously added, indicating a one-to-one relationship between the metals
0
5
89
II
10
12
Fig. 1. Abscissa: time (min); ordinare: OD. Effects of monovalent or divalent cations and ATP on the turbidity of axoneme suspensions (Anrhocidavis). 20 mM Tris-HCI buffer, pH 7.5; 0.44 mg protein/3 ml; 0.5 mM monovalent or divalent cations; 0.66 mM ATP.
and the chelating agents. It was found that the depressing effect of these reagents when Zn2+ or Cd2+, for instance, were used as the cations, diminished in the following order: phosEDTA > pyrophosphate > nucleoside phates. When Hg2+ was added, however, the once-elevated turbidity of the suspensions was only slightly reduced by adding ATP. This might be due, at least in part, to the strong affinity between this metal and the flagellar proteins. A similar, although less marked. result was obtained in the case of Fez+. As shown in fig. 2, ADP was also effective in reducing the turbidity increase due to either Zn2+ or Cd2+, but its effect was about half that of ATP. AMP was almost without effect. The same was true of the guanosine phosphates GTP, GDP and GMP. GTP as well as ITP and CTP decreased the turbidity to almost the same extent as ATP. The effect of UTP, however, was less than those of the other nucleoside triphosphates. Thus, there was found a parallelism between the depressing effect and the strength of chelating action. Exptl Cell Res 83 (1974)
90
M. Morisawa & H. Mohri
Fig. 3. Abscissa: time (mm); ordinate:
OD. Fluctuations in turbidity of axoneme suspensions induced by zinc or cadmium (Pseudocenfrotus). 20 mM Tris-HCI buffer, pH 8.2; 0.44 mg protein/3 ml; 0.05 mM ZnC1, or CdCI, were added at time 0.
Fluctuations in turbidity When the effect of several concentrations of Cd*+ on the turbidity of the microtubule suspensions was examined, it was found that at Cd2+ concentrations around 0.05 mM the turbidity alternately increased and decreased with a periodicity of a few minutes. The fluctuation gradually subsided and the turbidity finally reached a certain level dependent on the amount of metal added. As shown in fig. 3, the same was true with the suspensions of axonemes. Zinc as well as cadmium caused fluctuations in turbidity. Under the experimental conditions examined, the periodical change appeared most markedly at protein concentrations of about 0.15-0.2 mg/ml, suggesting a stoichiometric relationship between the added metal and the flagellar protein in producing the observed changes in turbidity. In these experiments, cadmium was used because it belongs to the same column as zinc on the periodic table, and was the most effective in increasing the turbidity of the suspensions. As described above, mercury, another metal in the same column, combined Exptl Cell Res 83 (1974)
irreversibly with the flagellar protein. Of these three metals, only zinc was found in flagella in considerable amounts [14]. Iron, which was also found in flagella, was not effective in causing the fluctuation in turbidity If ATP was added to the suspensions of axonemes (fig. 4) the turbidity of which had become stable after the initial addition of Zn2+. the fluctuations of turbidity reappeared. In this case, also, a one-to-one relationship was found between Zn2+ and ATP, i.e.. 0.05 mM ATP was the most effective in the presence of 0.05 mM ZnCI,. The use of aged axonemes in this particular experiment resulted in the disappearance of the typical fluctuation pattern when Zn’+ alone was present (see below). Restoration of the fluctuations was also caused by GTP and ADP, but to a much lesser extent than that caused by ATP. AMP was not effective. In all these cases, agitation of the suspensions with a glass rod during the measurements of turbidity failed to alter the fluctuation patterns, nor could any heavy precipitates be seen in the suspensions. The fluctuations, therefore, cannot be ascribed to convection currents in the cuvettes.
Ln
ATP
5 I
Fig. 4. Abscissn: time (min); ordinate: OD. Restoration by ATP of the fluctuations in turbidity of axoneme suspensions (Anthocidoris). 20 mM, Tris-HCI buffer, pH 8.2; 0.44 mg protein/3 ml; 0.05 mM &Cl,; ATP cont.: I, 0.05 mM; 2, 0.10 mM; 3, 0.20 mM; 4, 0.30 mM; 5, 0.50 mM.
Heavy metals and sperm motility. II
‘ATP
(0)
35
3oo: I 0
.34
2
4
6
8
IO
12
14
16
‘ATP 11j
(b)
I 2
30
-2 0
2
4
6
8
IO
12
14
16
Fig. 5. Abscissu: time (min); ordinate: OD. Effects of incubation at various temperatures on the fluctuation patterns of axoneme suspensions (Anthociduris). 20 mM Tris-HCI buffer, pH 8.2; 0.44 mg protein13 ml; 0.05 mM ZnCl,; 0.05 mM ATP. to) incubation at 0°C; I, for 12; 2, 22; 3, 36 h; (b) heat treatment. I, 37 C, 60 min; 2, 0 C, 0 min; 3, 57 C 30 min; 4, IOO’C, 60 min.
Factors influencing fluctuations in turhiditj In the following experiments, the effects of various factors were examined on the fluctuation which reappeared after the addition of 0.05 mM ATP, as such a fluctuation could be observed more consistently than the initial fluctuation in the presence of 0.05 mM Zn”+ or Cd2+ alone. As shown in fig. 5a, when the axoneme suspensions were stored at 0°C for varying lengths of time, the amplitude and frequency of the fluctuation became smaller in proportion to the length of time. The same was true when the temperature for standing the suspensions was increased. It was further ernphasized from the turbidity pattern in heat-treated axoneme suspensions (fig. 5b) that thermal denaturation of the flagellar proteins results in decrease or disappearance of the fluctuations.
91
When the axoneme suspensions were sonicated, the optical densities before and after the addition of Zn2+ decreased progressively with increasing time of sonication, which disintegrates the flagellar structures. Furthermore, the intensity of the fluctuations in the presence of ATP was gradually diminished and no fluctuations were observed after 2 min sonication. A solution of axonemes dissolved in 8 M urea did not show any fluctuations in turbidity. When the dissolved axonemes were dialysed against DDW for 30 h to remove the urea, however, the fluctuation pattern could be observed, although the amplitude of fluctuation was smaller than that of the intact axoneme suspension (fig. 6). The difference between these two cases suggests that the flagellar structures, once disrupted by 8 M urea, might have been reconstituted to some extent during the course of dialysis (see also
VW. Bovine serum albumin, which was examined instead of flagellar axonemes and microtubules, failed to show the fluctuations in turbidity. The results of subjecting Gactin and F-actin from rabbit skeletal muscle and insect flight muscle to the conditions
03
.oor--.
L-
O
3
2
4
6
8
10
12
14
16
Fig. 6. Abscissa: time (mm); ordinate: OD. Effects of urea treatment on the fluctuation patterns of axoneme suspensions (Anthocidoris). The experimental conditions were the same as in fig. 5. The figure shows the fluctuation oatterns observed after the addition of ATP. I, control, dialysed against DDW for 30 h; 2, dissolved in 8 M urea and then dialysed against DDW for 30 h; 3, dissolved in 8 M
urea.
Exptl Cell Res 83 (1974)
92
M. Morisawa & H. Mohri
under which fluctuations in turbidity were observed with axonemes or microtubules were also negative. DISCUSSION From the results obtained in the foregoing experiments, the change in turbidity induced by divalent cations and chelating agents seems to be analogous to the shortening and elongation of the thread model prepared from sea urchin eggs. In fact, the order of efficiencies of the divalent cations in causing contraction of the thread mode1 is similar to the order in which they increase the turbidity of flagellar suspensions [ 151. Furthermore, ATP failed to induce contraction of the thread mode1 in the presence of K+ and Mg2+. Instead, the nucleotide caused elongation of the mode1 which had shortened on the addition of divalent cations [26]. It has been pointed out, in connection with the thread model, that some metal ions other than Cu2+ and Fe’+, such as Cd2+, Zn2+ and Hg2+, would be expected to combine directly with -SH groups, forming mercaptide [15]. Such a mechanism might well be responsible for the changes in turbidity of the flagellar suspensions in addition to electrostatic attraction, affecting intermicrotubular, intermolecular or intramolecular connections. Our previous paper [14] reported that zinc and iron are concentrated in sea urchin sperm flagella. However, iron was found to combine rather irreversibly with the flagellar components and did not induce the fluctuations in turbidity. The observation that zinc combines with the flagellar protein in some fashion that is reversible by ATP suggests that this metal plays role in spermatozoan motility. Further differences between zinc and iron in their effects on the conformation of flagellar proteins will be shown in a following paper [I 31. Exptl
Ceil Res 83 (1974)
The intial fluctuations induced by minute amounts of Zn2+ or Cd2+ would arise from interactions between the added metal ions and the nucleotides contained in these preparations. The subsequent gradual subsidence of the fluctuations would then be associated with the decrease in the amount of such nucleotides, which could affect the binding of the metal ions to the flagellar proteins. In fact, it has been reported that guanosine nucleotides are present in isolated flagella and microtubules [ 11, 19,251, while adenosine nucleotides are apt to be washed out during the course of preparation. Alternatively. the synchronism of some phenomenon underlying the fluctuations in turbidity, once established by adding the metal ions, might be gradually disturbed. The restoration of the periodical changes in turbidity by the addition of equivalent amounts of ATP indicates that both the metal ions and the nucleotides are indispensable for the oscillation. It is quite probable that the periodical fluctuations are ‘physiological’, because they gradually disappeared with the lapse of time at various temperatures and on treatment with heat, ultrasonic waves or urea; moreover, they seem to be specific for the flagellar components. The phenomenon would be an in vitro expression of the rhythmic wave motion of sperm flagella, although the frequency of the fluctuations observed was much less than that of actual flagellar movement. This could be interpreted as due to loosening of the tight coupling between the flagellar components or loss of some factor(s) essential for flagellar movement. Tn recent years, considerable information has been accumulated concerning the mode of movement of flagella and cilia on the one hand, and the properties of their components, flagellar ATPase (dynein) and microtubule protein (tubulin), on the other. The predominant hypothesis presented so far to account
Heavy metals and sperm motility. II for the basic mechanisms of flagellar movement is the sliding theory, which was originally applied to muscle contraction [17], although there is also another hypothesis, which assumes the occurrence of contraction and relaxation of flagellar microtubules or their active units [18, 251. More recently, Summers & Gibbons [20] have shown that active sliding occurs between groups of the outer doublet microtubules of trypsin-treated sea urchin sperm flagella in the presence of ATP and magnesium. In any case, it is unquestionable that the flagellar microtubules are bent with a certain curvature, either passively or actively. Since analyses of the wave motion of sperm flagella have indicated that it consists of circular arcs combined with straight lines [I], a certain structural unit (tubulin molecule?) on a fixed point of flagella or microtubules would be expected to oscillate between states corresponding to ‘bend’ and ‘unbend’. The bending would be associated with changes in the interactions among flagellar structures and/or some conformational changes of the flagellar proteins. It can be assumed that the continuous combination and removal, by ATP, of zinc ions at certain binding sites on the flagellar proteins would cause cyclic changes in their conformation, resulting in the fluctuations in turbidity as an expression of such periodical changes at the macroscopic level. That the oscillation in the turbidity could be observed with a fraction containing only microtubules implies that the phenomenon takes place in the absence of dynein ATPase and is probably due to an intrinsic property of the microtubule protein. Since, however, the chelating activity of ADP is much less than that of ATP, the action of dynein ATPase would allow the zinc ions once released by ATP to recombine with the microtubule proteins, thus exaggerating the periodical changes.
93
In the present experiment, electron microscopical observations appeared to suggest that the periodical increase and decrease in turbidity might be due to aggregation and disaggregation of the flagellar microtubules. It is quite possible, however, that the changes in turbidity induced by heavy metals and chelating agents, especially zinc and ATP, would reflect not only the mutual aggregation or disaggregation of flagellar components, but also conformational changes in the flagellar proteins. Some promising results which have been obtained in this connection will be reported in a forthcoming paper
u31. The authors wish to thank Dr J. C. Dan for her kind help in preparing the manuscript. They are also indebted to Miss M. Shimomura for her technical assistance.Finally, the authors expresstheir gratitude to ProfessorH. Sugawarafor his kind interest and warm encouragement. This work was supported by grants-in-aid from the Ministry of Education, Japan, and grants (No. M67.0140, No. M69.0130, No. M71.08C, No. M72.051C)from the Population Council, Inc., USA. REFERENCES 1. Brokaw, C J, J exptl biol 43 (1965) 155. 7 Fujii, T, Annot zool japon 27 (1954) 115. 5: - J fat sci univ Tokyo IV 7 (1953) 224. 4. - Ibid IV 7 (1955)327. 5. Fujii, T, Utida, S, Mizuno, T & Nanao, S, J fat sci univ Tokyo IV 7 (1955)335. 6. Gibbons, I R, Arch biol (Littge) 76 (1965)317. 7. - J cell biol 26 (1965)707. 8. Lowry. 0 H, Rosebrough, N J, Farr, A L & Randall, R J,‘J biol them 193 (1951) 265. 9. Mohri, H, Biol bull 127(1964)381. IO. Mohri, H, Murakami, ‘s &’ Maruyama, K, J biochem 61 (1967)518. I I. Mohri, H & Yanagisawa,T, Zoo1mag 76 (1967)
276. 12. Morisawa, M, Zoo1mag 78 (I 969)484. 13. - In ureoaration. 14. Morisawa, M & Mohri, H, Exptl cell res 70 (1972) 311. 15. Sakai, H, J gen physiol 45 (1962)412. 16. - Personalcommunication. 17. Satir, P, J cell biol 39 (1968) 77. 18. Silvester, N R & Holwill, M E J, Nature 205 (I 965) 665. 19. Stephens, R E, Renaud, F L & Gibbons, I R,
Science156(1967)1606.
20. Summers, K E & Gibbons, I R, Proc natl acad sci US 68 (1971) 3092. Exptl Cell Res 83 (1974)
94
M. Morisawa
& H. Mohri
21. Tibbs, J, Spermatozoan motility (ed D W Bishop) p. 233. Am Ass Adv Sci, Washington, D.C. (1962). 22. - Nature 193 (1962) 686. 23. - Biochem j 89 (1963) 97. 24. - Ibid 96 (1965) 340.
Exptl Cell Res 83 (1974)
25. Yanagisawa, T, Hasegawa, S & Mohri, H, Exptl cell res 52 (1968) 86. 26. Yazaki, I. Personal communication. Received July 10, 1973