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Fine-structural localization of ATPase activity in mature sperm of Drosophila melanogaster
163 FINE-STRUCTURAL MATURE W. Laboratory
SPERM TH. for
DAEME,
LOCALIZATION OF ATPASE ACTIVITY OF DROSOPHILA MELANOGASTER J.-P.
PERSIJN
and
A. D.
TATES
Electron Microscopy and Department of Radiation University of Leiden, The Netherlands Received
IN
Genetics,
June 27,1963
NumRous studies
have demonstrated a clear-cut relationship between the splitting of ATP and the contraction of muscle. A similar relationship has been suggestedfor sperm-cell motility [5]. In both muscle [Ill and sperm [4] the ATP-splitting activities are thought to be connected with the contractile proteins themselves. In heart muscle, histochemically demonstrable ATPase activities were found to have characteristic and reproducible localizations [I]. It remained difficult, however, to relate the lead phosphate precipitate, indicating the location of enzyme activity, to any structural component of the muscle cells. With regard to sperm cells, the situation seemedmore promising: the tails of these cells, in which the contractile proteins are situated, are built up with a very characteristic structure, generally consisting of one or two central fibres and nine paired peripheral fibres. The latter are generally considered to be the contractile elements of sperm cells. As from muscle cells, a contractile protein possessingATPase activity could be extracted from sperm tails. This protein was called spermosin, and showed some similarity to myosin [2]. In order to determine the relationship between the location of ATPase activity and the above-mentioned fine-structural components of sperm tails, a number of investigations were carried out on mature sperm of Drosophila melanogaster, the preliminary results of which are given in this note. Results.-The histochemical experiments were based on a morphological study of the fine-structural characteristics of spermatogenesisin the fruit fly Drosophila melanogaster, a detailed description of which will be given elsewhere.Sections through mature sperm tails reveal two major components, one being the axial fibre complex and the other the paracrystalline helmet-shaped remnant of the “Nebenkern” (Fig. 1). In the axial fibre complex (Text Fig. l), two central fibres can be observed; just outside these, nine extremely densesecondary fibres can be distinguished. The latter are connected with nine peripheral fibres by “spokes” of moderate density. Seemingly attached to, but outside, these peripheral fibres, nine paired structures or “satellites” can be observed whose position with regard to the peripheral fibres is constant. In between the satellites, an “intersatellite space” is present. EXPERIMENT I: Testes of the fruit flies, after a short (I’) pre-fixation in osmium tetroxide, were incubated in a medium containing ATP and lead [lo]. After 30 min incubation at room temperature, the tissue was post-fixed in 0~0, (60 min) and after dehydration embedded in Epon 812. Ultrathin sections were stained with leadhydroxide [71. The axial fibres proved to have withstood the preparative procedures rather well: Experimental
Cell
Research
32
W. Th. Daems,
J.-P.
Persijn
and A. D. Tates
in cross-sections the different components of the axial fibre complex can still be clearly distinguished (Fig. 2). In addition, however, a maximum of nine lead granules can be observed in the intersatellite spaces. The location of these lead granules is constant. In the central part of the axial fibre complex, a lead precipitate can also be observed; this is, however, less constant in its location and cannot be correlated with any special part of the axial fibre complex.
Text-Fig. l.-Diagrammatic cross-section through the axial fibre complex of a mature perm cell of Drosophila melanogaster. For description see text. Numbers indicate the intersatellite spaces.
Lonaitudinal sections through the axial fibre complex likewise reveal both types ofipreipitate: (a) the peripheral one consists of linearly oriented granules, having a spacing that sometimes exceeds the thickness of an ultrathin section which explains the fact that less than nine granules are sometimes met within one cross section; (b) the irregular precipitate in the central area of the axial fibre complex. Precipitate was seldom observed outside the axial fibre complex. EXPERIMENT II: As a control for experiment I, after pre-fixation (I’) testes were
incubated for 30 min at room temperature in the incubation medium of experiment I but without ATP. Further preparations were performed as in experiment I. Remarkably, a lead precipitate was observed whose location appeared to be identical to that
obtained in experiment I. With
respect
to this
result,
two
possibilities
were
considered:
the precipitate
is
Figs. l-4.-Cross-sections through tails of mature sperm cells of Drosophila melanogaster. Fig. l-The two central fibres of the axial fibre complex can be clearly distinguished. Between these and the peripheral fibres, the extremely dense secondary fibres are visible. The numbered arrows indicate the intersatellite spaces. x 154,000. H indicates the helmet-shaped remnant of the Nebenkern.
Fig. 2.-Specimen from experiment I, incubated in a medium with ATP. The intersatellite spaces which clearly contain a lead precipitate are numbered. x 208,000. Fig. 3.-Specimen from experiment III: second incubation without ATP. The intersatellite spaces are devoid of lead precipitate. Note the increase in central precipitate as compared to Fig. 2. x 208,000.
Fig. 4.-Specimen from experiment clearly containing a lead precipitate compared to Fig. 2. x 208,000. Experimental
Cell Research
32
IV: second incubation with ATP. The intersatellite spaces are numbered. Note the increase in central precipitate as
A TPase
in sperm cells
165
Experimental
Cell Reseurch
32
Th. Daems,
W.
J.-P.
Persijn
and A. D. Tates
either a-specific, because it is not influenced by the omission of ATP, or is the result of splitting by the cell of its own ATP. To test these possibilities the following experiments were performed. EXPERIMENT III: If in the medium of experiment II a spontaneous splitting by the cell of its own ATP can occur, it might be expected that incubation would diminish or even exhaust the ATP reserve of the sperm cells. If during such incubation no TABLE
I.
Presence of A TP and/or
lead in the incubation
media.
Results
Experiment
1st incubation
I II III I\’
ATP Pb No Pb No No No No
ATP ATP Pb ATP Pb
2nd incubation
Intersatellite precipitate
Central precipitate
-
+
-I
-
+
AI
No ATP Pb ATP Pb
-
++
+
++
lead is available, the liberated phosphate ions are not precipitated. If then, after the incubation, the cells are brought into a similar medium containing lead (see Table I), the decreaseof the ATP reserve would be demonstrated by the diminution or absenceof the lead precipitate. Testes were pre-fixed (l’), incubated as indicated in Table I, and post-fixed. The results of this experiment showed that the intersatellite precipitate did not appear (Fig. 3); the quantity of the central precipitate, however, was greater than that of experiment II, and lead granules could also be observed outside the axial fibres. EXPERIMENT IV: If it is correct to explain the results of experiment III as exhaustion by the cell of its own ATP, it might be expected that addition of ATP from the outside would give rise to a re-appearance of the characteristic lead precipitate as observed in experiment I. Therefore, an experiment similar to experiment III was performed with only one difference: ATP was added to the second incubation medium (see Table I). In this experiment the intersatellite precipitate was again observed (Fig. 4). The amount of central precipitate was increased, as in the preceding experiment, and a rather large number of lead granules were observed outside the axial fibre complex. Concluding remarks.-In these experiments two types of lead precipitate were distinguished: a peripheral and a central type, showing dissimilar behavior in the individual experiments. The following remarks can be made. The most likely interpretation of the results of experiment II, in which the intersatellite precipitate appeared to be present even when the testes were incubated in a Experimental
Cell Research 32
A TPase in sperm cells
167
medium without ATP, is that the cell splits its own ATP. Support for this assumption can be found in the results of a biochemical analysis of mature sperm which demonstrated a considerable amount of ATP to be present in these cells [3]. Conclusive evidence is provided by the results of experiments III and IV, which clearly demonstrate that the intersatellite precipitate is dependent on the presence of ATP. In contrast to the intersatellite precipitate, the central precipitate is present in all experiments. It is remarkable that both experiments III and IV show an increased quantity of the central precipitate as compared to experiments I and II. This increase clearly appears to be independent of the addition of ATP to the second incubation medium (exp. IV). It thus seems unlikely that it is the result of a central ATP-ase activity. The increase, however, can be explained by a splitting by the cells of their own ATP during the first incubation: since no lead is present, the enzymatically liberated phosphate ions are able to diffuse away from the peripherally located sites of ATPase activity to the central part of the axial fibre complex. Later incubation with a lead-containing medium gives rise to precipitation of these phosphate ions, but now more or less at random. In preliminary experiments in which the first and second incubations were performed in the presence of PCMB, which has been reported to inhibit ATPase activity [9], this increase of the central precipitate could indeed be prevented. From the experiments performed thus far it can be deduced that in the sperm cells of Drosophila melanogaster the ATPase activity is located in the intersatellite spaces, outside the peripheral filaments. This is in agreement with the observations of Lansing and Lamy [6], who, in the cilia of a rotifer, also demonstrated ATPase activity to be present outside the peripheral filaments. Our results are, however, in disagreement with those of Nelson [S], who, on the basis of a different technique, supposes the ATPase activity of mature sperm cells to be located in the peripheral fibres. Further studies are being devoted to this problem, dealing with substrate specificity of the enzymatic reaction described here. REFERENCES 1. DE BEYER, J. M., DE MAN, J. C. H. and PERSIJN, J.-P., J. Cell Biof. 13, 452 (1962). 2. BURNASHEVA, S. A., Biokhimia 23, 558 (1958). 3. ENGELHARDT, V. A., Advances in Enzymol. 6, 147 (1946). 4. ENGELHARDT; V. A..and BURNASHEV~, S. A., Biokhimia 22,554 (1957). 5. FAWCETT. D. W.. The Cell, vol. 2, n. 217. Academic Press, Inc., New York, 1961. 6. LANSING,‘A. I. and LAMY,‘F., .7. b>ophys. Biochem. Cyfol..ll, 498 (1961). 7. MILLONIG, G., J. Biophys. Biochem. Cyfof. 11, 736 (1961). 8. NELSON, L., Biochim. Biophys. Acfa 27, 634 (1958). 9. PADYKULA, H. A. and HERMAN, E., J. Hisfochem. Cyfochem. 3, 170 (1955). 10. PERSIJN, J.-P., DAEMS, W. TH., DE MAN, J. C. H. and MEYER, A. E. F. H., Histochemie 11.
372 (1961). SZENT-GY~RGYI, A., Chemical demic Press Inc., New
Physiology York, 1953.
of Contraction
in
Body
and
Experimental
Heart
Muscle.
Cell
Research
2, Aca-
32
SPERM TH. for
DAEME,
LOCALIZATION OF ATPASE ACTIVITY OF DROSOPHILA MELANOGASTER J.-P.
PERSIJN
and
A. D.
TATES
Electron Microscopy and Department of Radiation University of Leiden, The Netherlands Received
IN
Genetics,
June 27,1963
NumRous studies
have demonstrated a clear-cut relationship between the splitting of ATP and the contraction of muscle. A similar relationship has been suggestedfor sperm-cell motility [5]. In both muscle [Ill and sperm [4] the ATP-splitting activities are thought to be connected with the contractile proteins themselves. In heart muscle, histochemically demonstrable ATPase activities were found to have characteristic and reproducible localizations [I]. It remained difficult, however, to relate the lead phosphate precipitate, indicating the location of enzyme activity, to any structural component of the muscle cells. With regard to sperm cells, the situation seemedmore promising: the tails of these cells, in which the contractile proteins are situated, are built up with a very characteristic structure, generally consisting of one or two central fibres and nine paired peripheral fibres. The latter are generally considered to be the contractile elements of sperm cells. As from muscle cells, a contractile protein possessingATPase activity could be extracted from sperm tails. This protein was called spermosin, and showed some similarity to myosin [2]. In order to determine the relationship between the location of ATPase activity and the above-mentioned fine-structural components of sperm tails, a number of investigations were carried out on mature sperm of Drosophila melanogaster, the preliminary results of which are given in this note. Results.-The histochemical experiments were based on a morphological study of the fine-structural characteristics of spermatogenesisin the fruit fly Drosophila melanogaster, a detailed description of which will be given elsewhere.Sections through mature sperm tails reveal two major components, one being the axial fibre complex and the other the paracrystalline helmet-shaped remnant of the “Nebenkern” (Fig. 1). In the axial fibre complex (Text Fig. l), two central fibres can be observed; just outside these, nine extremely densesecondary fibres can be distinguished. The latter are connected with nine peripheral fibres by “spokes” of moderate density. Seemingly attached to, but outside, these peripheral fibres, nine paired structures or “satellites” can be observed whose position with regard to the peripheral fibres is constant. In between the satellites, an “intersatellite space” is present. EXPERIMENT I: Testes of the fruit flies, after a short (I’) pre-fixation in osmium tetroxide, were incubated in a medium containing ATP and lead [lo]. After 30 min incubation at room temperature, the tissue was post-fixed in 0~0, (60 min) and after dehydration embedded in Epon 812. Ultrathin sections were stained with leadhydroxide [71. The axial fibres proved to have withstood the preparative procedures rather well: Experimental
Cell
Research
32
W. Th. Daems,
J.-P.
Persijn
and A. D. Tates
in cross-sections the different components of the axial fibre complex can still be clearly distinguished (Fig. 2). In addition, however, a maximum of nine lead granules can be observed in the intersatellite spaces. The location of these lead granules is constant. In the central part of the axial fibre complex, a lead precipitate can also be observed; this is, however, less constant in its location and cannot be correlated with any special part of the axial fibre complex.
Text-Fig. l.-Diagrammatic cross-section through the axial fibre complex of a mature perm cell of Drosophila melanogaster. For description see text. Numbers indicate the intersatellite spaces.
Lonaitudinal sections through the axial fibre complex likewise reveal both types ofipreipitate: (a) the peripheral one consists of linearly oriented granules, having a spacing that sometimes exceeds the thickness of an ultrathin section which explains the fact that less than nine granules are sometimes met within one cross section; (b) the irregular precipitate in the central area of the axial fibre complex. Precipitate was seldom observed outside the axial fibre complex. EXPERIMENT II: As a control for experiment I, after pre-fixation (I’) testes were
incubated for 30 min at room temperature in the incubation medium of experiment I but without ATP. Further preparations were performed as in experiment I. Remarkably, a lead precipitate was observed whose location appeared to be identical to that
obtained in experiment I. With
respect
to this
result,
two
possibilities
were
considered:
the precipitate
is
Figs. l-4.-Cross-sections through tails of mature sperm cells of Drosophila melanogaster. Fig. l-The two central fibres of the axial fibre complex can be clearly distinguished. Between these and the peripheral fibres, the extremely dense secondary fibres are visible. The numbered arrows indicate the intersatellite spaces. x 154,000. H indicates the helmet-shaped remnant of the Nebenkern.
Fig. 2.-Specimen from experiment I, incubated in a medium with ATP. The intersatellite spaces which clearly contain a lead precipitate are numbered. x 208,000. Fig. 3.-Specimen from experiment III: second incubation without ATP. The intersatellite spaces are devoid of lead precipitate. Note the increase in central precipitate as compared to Fig. 2. x 208,000.
Fig. 4.-Specimen from experiment clearly containing a lead precipitate compared to Fig. 2. x 208,000. Experimental
Cell Research
32
IV: second incubation with ATP. The intersatellite spaces are numbered. Note the increase in central precipitate as
A TPase
in sperm cells
165
Experimental
Cell Reseurch
32
Th. Daems,
W.
J.-P.
Persijn
and A. D. Tates
either a-specific, because it is not influenced by the omission of ATP, or is the result of splitting by the cell of its own ATP. To test these possibilities the following experiments were performed. EXPERIMENT III: If in the medium of experiment II a spontaneous splitting by the cell of its own ATP can occur, it might be expected that incubation would diminish or even exhaust the ATP reserve of the sperm cells. If during such incubation no TABLE
I.
Presence of A TP and/or
lead in the incubation
media.
Results
Experiment
1st incubation
I II III I\’
ATP Pb No Pb No No No No
ATP ATP Pb ATP Pb
2nd incubation
Intersatellite precipitate
Central precipitate
-
+
-I
-
+
AI
No ATP Pb ATP Pb
-
++
+
++
lead is available, the liberated phosphate ions are not precipitated. If then, after the incubation, the cells are brought into a similar medium containing lead (see Table I), the decreaseof the ATP reserve would be demonstrated by the diminution or absenceof the lead precipitate. Testes were pre-fixed (l’), incubated as indicated in Table I, and post-fixed. The results of this experiment showed that the intersatellite precipitate did not appear (Fig. 3); the quantity of the central precipitate, however, was greater than that of experiment II, and lead granules could also be observed outside the axial fibres. EXPERIMENT IV: If it is correct to explain the results of experiment III as exhaustion by the cell of its own ATP, it might be expected that addition of ATP from the outside would give rise to a re-appearance of the characteristic lead precipitate as observed in experiment I. Therefore, an experiment similar to experiment III was performed with only one difference: ATP was added to the second incubation medium (see Table I). In this experiment the intersatellite precipitate was again observed (Fig. 4). The amount of central precipitate was increased, as in the preceding experiment, and a rather large number of lead granules were observed outside the axial fibre complex. Concluding remarks.-In these experiments two types of lead precipitate were distinguished: a peripheral and a central type, showing dissimilar behavior in the individual experiments. The following remarks can be made. The most likely interpretation of the results of experiment II, in which the intersatellite precipitate appeared to be present even when the testes were incubated in a Experimental
Cell Research 32
A TPase in sperm cells
167
medium without ATP, is that the cell splits its own ATP. Support for this assumption can be found in the results of a biochemical analysis of mature sperm which demonstrated a considerable amount of ATP to be present in these cells [3]. Conclusive evidence is provided by the results of experiments III and IV, which clearly demonstrate that the intersatellite precipitate is dependent on the presence of ATP. In contrast to the intersatellite precipitate, the central precipitate is present in all experiments. It is remarkable that both experiments III and IV show an increased quantity of the central precipitate as compared to experiments I and II. This increase clearly appears to be independent of the addition of ATP to the second incubation medium (exp. IV). It thus seems unlikely that it is the result of a central ATP-ase activity. The increase, however, can be explained by a splitting by the cells of their own ATP during the first incubation: since no lead is present, the enzymatically liberated phosphate ions are able to diffuse away from the peripherally located sites of ATPase activity to the central part of the axial fibre complex. Later incubation with a lead-containing medium gives rise to precipitation of these phosphate ions, but now more or less at random. In preliminary experiments in which the first and second incubations were performed in the presence of PCMB, which has been reported to inhibit ATPase activity [9], this increase of the central precipitate could indeed be prevented. From the experiments performed thus far it can be deduced that in the sperm cells of Drosophila melanogaster the ATPase activity is located in the intersatellite spaces, outside the peripheral filaments. This is in agreement with the observations of Lansing and Lamy [6], who, in the cilia of a rotifer, also demonstrated ATPase activity to be present outside the peripheral filaments. Our results are, however, in disagreement with those of Nelson [S], who, on the basis of a different technique, supposes the ATPase activity of mature sperm cells to be located in the peripheral fibres. Further studies are being devoted to this problem, dealing with substrate specificity of the enzymatic reaction described here. REFERENCES 1. DE BEYER, J. M., DE MAN, J. C. H. and PERSIJN, J.-P., J. Cell Biof. 13, 452 (1962). 2. BURNASHEVA, S. A., Biokhimia 23, 558 (1958). 3. ENGELHARDT, V. A., Advances in Enzymol. 6, 147 (1946). 4. ENGELHARDT; V. A..and BURNASHEV~, S. A., Biokhimia 22,554 (1957). 5. FAWCETT. D. W.. The Cell, vol. 2, n. 217. Academic Press, Inc., New York, 1961. 6. LANSING,‘A. I. and LAMY,‘F., .7. b>ophys. Biochem. Cyfol..ll, 498 (1961). 7. MILLONIG, G., J. Biophys. Biochem. Cyfof. 11, 736 (1961). 8. NELSON, L., Biochim. Biophys. Acfa 27, 634 (1958). 9. PADYKULA, H. A. and HERMAN, E., J. Hisfochem. Cyfochem. 3, 170 (1955). 10. PERSIJN, J.-P., DAEMS, W. TH., DE MAN, J. C. H. and MEYER, A. E. F. H., Histochemie 11.
372 (1961). SZENT-GY~RGYI, A., Chemical demic Press Inc., New
Physiology York, 1953.
of Contraction
in
Body
and
Experimental
Heart
Muscle.
Cell
Research
2, Aca-
32