The thermal denaturation of nucleoprotein in boar sperm

J. Mol. Biol. (1965) Il, 1-11

The Thermal Denaturation of Nucleoprotein in Boar Sperm PETER

J.

CHAMBERLAIN AND PETER

M. B.

WALKER

Zoology Department, University of Edinburgh, Scotland (Received 30 December 1963, and in revised form 9 July 1964) The paper describes a study of the thermal denaturation of DNA in intact boar sperm. A recently designed microspectrophotometer was used to measure the integrated absorption at 260 mfL of sperm after they had been heated in several media. Denaturation was detected with heating media containing 70% glycerol and 30 % of a sodium chloride-sodium citrate solution. In addition, small changes in the integrated absorption at temperatures lower than those used for total thermal denaturation were seen. The possible significance of these observations is discussed.

1. Introduction The problem of control mechanisms in the differentiation of higher cells is beginning to focus attention on the state of the DNA in the nuclei of mammalian cells. The techniques available for such studies are severely restricted and this paper describes experiments in which the thermal denaturation of the nucleic acids in intact sperm has been investigated by measuring absorbance changes. It was hoped that by comparing these results with those for free nucleic acids in solution, information could be obtained on the bonds restraining the nucleic acids within cells. Mammalian sperm were chosen for several reasons. They contain a constant and eeproducible amount of DNA such that the microspectrophotometer used could .Jv:"et no significant differences between the absorbancies of individual sperm. Also . / contain a negligible amount of RNA; for instance, bull sperm have been shown to contain less than 0·02% RNA (Abraham & Bhargava, 1963). In addition, when in media of high refractive index, their optical properties allow accurate measurements to be made of the amount of DNA without any pretreatment with cytochemical fixatives (Walker, 1957). In these experiments we have used a recently designed microspectrophotometer, (Walker, Leonard, Gibb & Chamberlain, 1963), which measures a parameter termed the "integrated absorbance" of individual sperm. This quantity is the sum of the absorbances at 260mfL recorded through a l'fL diameter aperture which is moved over the complete area of the sperm. By using this technique the most serious errors inherent in measurements made with earlier microspectrophotometers can be greatly reduced.

2. Materials and Methods Fresh boar semen was supplied by the Animal Breeding Research Station, Roslin, Midlothian and stored at - 80 0 e after adding 10% glycerol. Some of the frozen material was diluted with saline-citrate, SSet, and washed 3 times by centrifuging in the same medium. The washed cells were resuspended in sse and glycerol was then added to give a final concentration (by volume) of 70% glycerol. Separate portions were heated in a tube t A solution of x M-saline--citrate contains x M·sodium chloride and 0·1 x M·sodium citrate and a solution of x M·SSC/G contains 30% x M·SSC + 70% glycerol. 1

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P. J. CHAMBERLAIN AND P. M. B. WALKER

placed in a thermostatically controlled water-bath or in a xylene-benzene mixture boiling under reflux. At the appropriate time a drop of the heated suspension was placed between quartz slides at room temperature for measurement. The method of measurement is fully described in Walker et al, (1963), and consists of summing the absorbances at 260 mJL from each element 1 JL in diameter in a two-dimensional scanning pattern over the entire sperm head. The resulting instrument reading is called the integrated absorbance (E A;J. Later measurements (Figs 5 and 6) requiring a higher degree of accuracy were made on a modification of the original instrument, in which the specimen and reference beams were monitored by separate photomultipliers, and in which the mechanics of the condenser and objective slides had been considerably improved. Each point recorded on the denaturation curves is the mean of four separate integrations from each of eight different sperm, and the standard deviation of these eight individual means from a combined mean of 3·00 was typically 0·07. Boar sperm DNA was prepared by a modification of the method described by Borenfreund, Fitt & Bendich (1961). Sperm were washed twice in 0·15 M-SSe, then suspended in 0·15 M-SSe containing 5% by volume of 2.mercaptoethanol and gently stirred for 1 hr at 4°e. Trypsin at 0·5 mgjml, was added, the pH adjusted to 7·8 and stirring continued for a further hour at room temperature. Duponol at 0'5% and sodium p.aminosalicylate at 6% were added and the solution stirred for 15 min before finally adding an equal volume of phenol saturated with 0·3 M-sodium trichloroacetate solution and stirring for a further 30 min. The aqueous phase was then separated from the phenol by centrifugation and treated twice more with fresh phenol. It was then washed 4 times with ether and the nucleic acid "spooled" out on a glass rod after the addition of an equal volume of ethanol. The nucleic acid was then dissolved in 1·5 X 10- 3 M·SSe. A nucleohistone extract was prepared by lysing the sperm after first gently oxidizing the -S-S- bonds with sodium peroxyacetate. Sperm were washed twice in 0·15 M·SSe and then suspended in a solution containing 0·15 M-sodium peroxyacetate plus 0·15 M.sodium acetate, pH 7·4, obtained by back-extracting a solution of the corresponding acids in ether with 0·3 M·sodium bicarbonate solution. The ether solution was prepared by the method of Stahmann & Bergmann (1946). The suspension of sperm was stirred for 1 hr at room temperature, after which the sperm were washed once with 0·15M-SSe, then several times with 1·5 X 10- 3 M-SSe. After washing in this solution the sperm heads swell to about 30 times their original volume although still maintaining their attachment to the middle pieces. A suspension of this "gel" of sperm, after dilution with 70% glycerol and suitable adjustment of the salt concentration, was used in the melting point studies. The ultraviolet absorbing material extracted from sperm heads was determined in the following way. Semen was washed as described, and the suspension extracted with 0·15 M-SSe/G for various times and temperatures. The extracted suspension was then centrifuged at 4°e in a Spinco model L ultracentrifuge at 40,000 g for 60 min. The super· natant fraction was divided into two further Spinco tubes, diluted twofold with 0·15 M· sse and recentrifuged as before. The absorbance at 260 mJL of the final supernatant solution was then measured. This absorbance was compared with that from the material extracted in 20 min at 700 e by 9% perchloric acid from another equal sample of washed sperm. For high-temperature measurements on intact sperm, specially designed glycerol immersion reflecting objectives were used (R. & J. Beck Limited, London). The condenser, objective and specimen slide were surrounded with glycerol contained in a copper waterjacket and joined to the condenser with a flexible rubber seal. Hot water was pumped through the copper jacket and the glycerol was responsible for conveying heat to the specimen slide. A thermocouple was used to relate the temperature of the hot water to that of the specimen.

3. Results (1) Denaturation in situ Early experiments in which either sucrose or liquid paraffin was used to increase the refractive index of the medium in which the sperm were suspended, failed to show any convincing increase in the integrated absorbance of cooled cells which had been

DENATURATION IN BOAR SPERM

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heated to temperatures up to 100°0. However, when a suspension of sperm in 70% glycerol was heated for 30 minutes at 105°0 an increase of some 30% in the integrated absorption was found. Figure 1 shows the complete curve obtained when the heating 70% glycerol). medium was 0·15 M-SSO/G (30% 0·15 M-SSO

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FIG. 1. Sperm suspended in 70% glycerol and 30% 0·15 M-SSC. The cells were heated for 1 hr at the temperature stated before cooling and measuring, except above 100°C, when this time was reduced to 30 min.

In order to be sure that the increased absorption after treatment at 105°0 is caused by a genuine increase in the specific nucleic acid absorption and not an increase in light-scattering by proteins, spectra were recorded through the heads of both heated and unheated sperm. Both spectra showed A240/A260 ratios of 0·6 and negligible absorptions at 320 mp.. Further confirmation that the increased absorption is the result of nucleic acid denaturation was found on extracting the DNA from both heated and unheated sperm. DNA from sperm treated for one hour either at 20 or 75°0 with the glycerol medium showed a 35% increase in absorption at 260 mp. when heated to 95°0 in the 0·15 M-SSO/G medium whilst DNA from cells heated to 110°0 for 30 minutes in the same medium showed no increase. In another experiment, very similar hyperchromic effects were found in DNA extracted from sperm after treatment in 0·15 M-SSO/G for one hour at 20,45,60 and 75°0. However, since it proved very difficult to extract DNA quantitatively from sperm once they had been heated in 0·15 M-SSO/G, it cannot be said that the small increase in absorbance at 60°0 of sperm (Fig. 1) is not caused by the denaturation of a portion of the DNA. (2) Boar sperm nucleohistone

Berry & Mayer (1960) have extracted a histone-like protein from bull sperm, and attempts were made to produce a soluble nucleohistone preparation from boar sperm so that results from the nucleohistone could be compared with those from intact sperm. This proved very difficult and eventually a gel of expanded sperm

P. J. CHAMBERLAIN AND P. M. B. WALKER

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suspended in the appropriate medium was used. The gel was prepared after gently oxidizing the -S-S- bonds of the sperm protein (see Materials and Methods). In SSC/G media the suspension had an A320/A260 ratio of 0·25 and an A240/A260 ratio of 1. After correcting for non-specific protein scatter by the method of Bonhoeffer & Schachman (1960), it was found that a T m of 72°C in 0·15 M-SSC/G was obtained whether absorption measurements were made on hot solutions, or after cooling them. This contrasts with the results found with DNA (Geiduschek, 1962). For example, with boar sperm DNA in 0·15 M-SSC/G, a T m of 60°C is increased to 69°0 if the solutions are cooled after heating and before taking absorbance measurements. (3) Effect of ionic strength on melting in sperm In addition to making integrated absorption measurements on sperm after heating in 0·15 M-SSC/G, media containing 0·03 M-SSC/G and 0·3 M-SSC/G were used. In both these, denaturation was observed and the melting point in all three media was found to be 92°0. This invariance is unlike the results observed with purified DNA (Marmur & Doty, 1962). However, when the boar sperm nucleohistone preparation was heated in the same three media, similar melting points of 72°0 were found. (4) Kinetics of denaturation

The time sequence of the denaturation process in intact sperm heated in 0·15 M· SSO/G at 106°0 is shown in Fig. 2. It can be seen that the maximum increase is found

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FIG. 2. Cells heated at 106°C for the times stated. Otherwise as for Fig. 1. The left-hand part demonstrates that, during the initial period of denaturation, first-order kinetics are followed.

after 20 minutes heating and this is followed by a decrease in absorption. During the initial heating the expected first-order kinetics are found. The slow reaction is quite unlike that found in purified DNA and boar sperm nucleohistone, when absorbance changes are almost instantaneous.

DENATURATION IN BOAR SPERM

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(5) Renaturation

The experiments of Marmur & Doty (1961) have shown that it is possible to "anneal" denatured bacterial and virus DNA but not mammalian DNA by heating just below the temperature at which "melting" starts. With the h)gh concentration of DNA in a sperm and the restriction imposed by the protein on movements of the DNA strands, it was thought that this system might be one in which it was possible to observe renaturation of mammalian DNA. Sperm were heated for 15 minutes at 106°0 in 0'15M-SSO/G, after which the suspension was maintained at 70°0 for up to 18 hours (Marmur & Doty, 1961). This caused the integrated absorption after cooling to be reduced by up to 12%. However, when the sperm were reheated for 10 minutes at 106°0, the absorption was further reduced, probably due to a loss of nucleic acid. We therefore conclude that renaturation has not occurred, and that the reduction after the prolonged incubation at 70°0 is probably due to loss of absorbing material from the sperm head. (6) Substitution of sucrose for glycerol

The early experiments mentioned, in which denaturation appeared impossible in sucrose solutions, have been investigated further. A 52% w/w solution of sucrose in 0·15 M-SSO was chosen since its refractive index matched that of 0·15 M-SSO/G and no correction for the optical path length was then required. The results (Fig. 3)

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FIG. 3. Comparison of glycerol and sucrose heating media. Conditions are those used in Fig. 1. The sucrose medium was 52% sucrose and 48% 0·15 M-SSO (w/w) and the 0·15 M.SSC/G curve was taken from Fig. 1.

show that denaturation cannot be seen at temperatures below 110°0. Fig. 3 also shows a rise at 70°0 similar to that found in 0·15 M-SSO/G at 60°0. However, in this case, it is of smaller height and is spread over a greater temperature range. The melting of the boar sperm nucleohistone in the same sucrose medium was also investigated. It showed a melting temperature of 79°0, some 7°0 higher than that

P. J. CHAMBERLAIN AND P. M. B. WALKER

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observed in the glycerol medium. Also, in this medium, as in the glycerol medium, the same melting temperature was found for both cooled and uncooled nucleohistone. (7) Ultraviolet-absorbing material extracted by SSCjG

Both Figs 1 and 3 show that small changes in the integrated absorption of sperm take place at temperatures lower than those used for total denaturation and it is possible that a loss of nucleic acid from the sperm could explain some of these. In order to investigate this possibility, the amount of material absorbing at 260 mp. which is extracted by 0·15 M-SSOjG was measured (Fig. 4). It had an A 2 6 0 which

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FIG. 4. The A 26 0 of 0·15 M.SSC/G extracted material, expressed as a percentage of the A 2 6 0 of material extracted by hot perchloric acid. Left, 1 hr heating in 0·15 M.SSC/G at the temperature stated. The results of several different experiments ate shown.

corresponded to between 5 and 10% of the material extracted by hot perchloric acid, but there appeared to be little difference between the amounts extracted at 105°0 and at 18°0. From this and an A240jA260 ratio approaching 0·9 in the extracts, it seems that much of the material is protein in origin and comes perhaps from the tails and middle-pieces. Extraction cannot therefore account for the shape of the denaturation curve. (8) The effects of short times of heating at temperatures between 50 and 70°C

The kinetics of the absorbance changes in 0·15 M-SSOjG were investigated, although this meant working at the limit of accuracy of the instrument despite the modifications mentioned in the Materials and Methods section. At temperatures at or just below the maximum of the small peak of absorptivity in the sperm heads, there is a progressive increase in the integrated absorption with time (Fig. 5). At temperatures just above this peak it is seen that there is an initial increase followed by a decrease. We have also investigated whether the increase in integrated absorption which occurs after heating at 60°0 can be reversed. Two separate experiments, in which the sperm were first heated for one hour at 60°0 and then for a further hour at 45, 60

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FIG. 5. (a) The effect of short times of heating on the integrated absorbance of sperm at temperatures between 55 and 75°0 in 0·15 M-SSO/G. (b) The result of heating for 1 hr at the stated temperature.

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FIG. 6. The effect of first heating sperm heads to 60°0 for 60 min and then heating for a further 60 min at the temperatures stated on the right.

or 75°0 are shown in Fig. 6. It will be seen first that, while further heating at 60°0 has little effect, treatment at 45 and 75°0 does cause some reduction in absorption and to this extent the increase in absorptivity at 60°0 may be reversible. (9) Sub-melting point effects at other ionic strengths

Heating curves were obtained for two other salt concentrations; in 0·03 M-SSOjG it was found that there was no consistent pattern in measurements below 75°0.

P. J. CHAMBERLAIN AND P. M. B. WALKER

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However, measurements made in 0·3 M-SSC/G revealed a more complex shape than that found in 0·15 M-SSC/G. A similar rise and fall was found between 60 and 75°C. However, the absorbance of unheated sperm was found to be 8% higher than in 0·15 M-SSC/G. This increase disappeared after heating between 50 and 60°C. (10) Absorption changes at different salt concentrations

In view of this unexpected result, the absorption of sperm at room temperature was investigated over a wider range of salt concentrations (Fig. 7). The curve has a

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FIG. 7. Effect of salt concentration on absorbance at room temperature. The point on the right s a measurement of sperm in 30% seminal fluid and 70% glycerol.

marked discontinuity at salt concentrations in the range 0·2 to 0·3 M.SSC/G. However, no similar discontinuity could be observed with the nucleohistone preparation under similar conditions. Figure 7 also shows that the integrated absorption increases at salt concentrations above 1 M in a fashion similar to DNA alone (Emmanuel, 1960). The reversibility of the transition from 0·2 M to 0·3 M-SSC/G was investigated. It was found that sperm first treated with 0·3 M-SSC/G and then suspended in 0·2 MSSC/G showed a decrease in absorption to a position midway between the usual 0·2 M and 0·3 M values. Parallel results were obtained for changes in the other direction. (11) The effect of glycerol on the melting point of DNA

Agents which reduce the dielectric constant of the solvent, like methanol (Geiduschek, 1962), glycol and glycerol (Duggan, 1961) also reduce the melting point of DNA. This effect was reinvestigated with media like those used for suspending sperm. With boar sperm DNA it was found that the melting point of 86°C in 0·15 MSSC was reduced to 61°Cin a solution of 0·15 M-SSC/G. As was mentioned earlier, the melting temperature of DNA is increased by some 9°C if the DNA samples are cooled before making absorbance measurements. In Fig. 8 the melting profiles of cooled sperm heads, DNA and nucleohistone and of uncooled DNA, all in 0·15 MSSC/G, are compared. It can be seen that the small rise at 60°C in the absorptivity of sperm occurs at a similar temperature to the melting of DNA.

9

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FIG. 8. Comparison of the thermal denaturation curves of boar sperm DNA, before (---) and after (-. -. -) cooling; and of nucleohistone (-0-0-) with sperm heads ( - - ) , all measured in 0·15 M-SSCjG.

(12) Measurements on sperm at high temperatures

Recently we have developed an apparatus which enables us to measure the integrated absorptions of sperm at temperatures up to 75°0 (see Materials and Methods). Using this apparatus it has been possible to confirm that observations made at high temperatures produce very similar results to those made on sperm after heating and then cooling to room temperature. In particular, it was found that the inconsistent results obtained with cooled sperm in 0·03 M-SSO/G were not a reflection of complete denaturation which was partially reversed on cooling. The profile in this medium, measured at high temperatures, showed no significant trend for temperatures up to 75°0.

4. Discussion The results in this paper show that the nucleohistone in sperm heads can be denatured in a glycerol medium if the temperature is taken high enough. In some ways this result is surprising, since it might be expected that with the very high concentration of nucleoprotein in these cells ( ",0,2 g/cm 3 ) the relative displacement of the strands by thermal agitation would be severely restricted. Indeed, since denaturation could be observed in a glycerol medium and not in a sucrose medium, it seems possible that a specific glycerol-nucleoprotein interaction is involved in the denaturation process . This view is supported by the observation that it is very much more difficult to extract DNA from sperm after they have been denatured in the glycerol medium. The intrinsic difficulty involved in denaturing DNA in an intact nucleus is illustrated by the work of Nash & Plaut (1964). They found that, in order to observe denatured DNA in the chromosomes of Drosophila salivary gland nuclei, the specimens had to be heated at 75°0 first for 20 minutes in water and then for 10 minutes in 4% formaldehyde solution.

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P. J. CHAMBERLAIN AND P. M. B. WALKER

In our experiments the unexpected effects in the temperature range 45 to 75°C seem of particular interest, but we have been unable to find a completely satisfactory explanation for them. Since there is no increased extraction of ultraviolet-absorbing material on heating sperm, the changes observed seem to be the result of molecular rearrangements. A comparison of the melting curves of DNA and nucleohistone in 0·15 M-SSC/G (Fig. 8) shows that the initial increase in the integrated absorbance at 60°C of the sperm heads can be attributed to the presence of a small part of DNA in the chromosome which is either protein-free or bound to protein in a manner different from the remainder. Bonner & Huang (1963) have also found that a "crude" extract of pea embryo chromatin shows a two-part melting profile. About a fifth of the total increase in absorbance in their preparation occurred over a temperature range similar to that of DNA, while the remainder behaved like nucleohistone. On the other hand, some interesting experiments by Felscnfeld, Sandeen & von Hippel (1963) suggest that DNA can form a complex with certain proteins which is more specific than that normally assigned to histones. They found that a mixture of native ribonuclease and DNA melted at a lower temperature than DNA alone. If the melted complex was heated to the temperature at which ribonuelease was denatured, the absorptivity decreased, and on further heating increased again to give a melting temperature some 20°C higher than that of DNA alone. The kinetics of our small changes at 60°C (Figs 5 and 6) are suggestive of the formation of some such complex between DNA and undenatured protein. However, in Felsenfeld's experiments the effect was more noticeable at low salt concentrations; this is not the case here. It may be significant that the changes in absorptivity which we find in the range 45 to 75°C are very similar in magnitude to the salt-induced changes at room temperature. The two effects may, therefore, be interrelated. These salt-induced changes are perhaps the most interesting finding of this paper since, as they are produced by relatively mild treatments, they may have physiological importance. They occur over the range of salt concentrations where the nucleohistone has a minimum solubility, and one explanation may be that they reflect conformational changes in the DNA similar to those found by Langridge et al. (1957) for DNA fibres at different humidities. These forms of DNA differ in helix pitch and in orientation of the bases to the axis, so that small differences in their absorptivities can be anticipated. In conclusion, it may be said that the results presented in this paper show that a relatively sophisticated spectroscopic analysis may be made of a biological system as complex as an intact sperm. Considerable difficulties arise when the analysis is extended to the nuclei of more functional cells, since these contain RNA in addition to DNA. Also, in growing cells the nuclei do not have a constant amount of DNA and the nucleic acids they do contain are likely to leak from the nuclei on heating. However, since we have developed the technique of measurement at high temperatures, heating curves can now be obtained by taking all the measurements from one cell. By surrounding such a cell with a non-aqueous phase such as liquid paraffin, problems of leakage can be overcome and so none of the difficulties mentioned appear insuperable. In addition, it seems that, with some modification, the heating apparatus can be made to work at temperatures of up to 1l0°C, which will greatly increase its usefulness.

DENATURATION IN BOAR SPERM

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We gratefully acknowledge most helpful comment and criticism from Drs S. T. Bailey, G. Felsenfeld, P. Geiduschek and A. R. Peacocke. Most of this work was undertaken while one of us (P. M. B. W.) held the Royal Society Research Fellowship in Cytochemistry. We would also like to acknowledge the generous financial support of the Royal Society and the Medical Research Council.

REFERENCES Abraham, K. A. & Bhargava, P. M. (1963). Biochem. J. 86, 298. Berry, R. E. & Mayer, D. T. (1960). Exp. Oell Res. 20, 116. Bonhoeffer, F. & Schachman, H. K. (1960). Biochem, Biophys. Res. Oomm. 2, 366. Bonner, J. & Huang, R-C. C. (1963). J. Mol. Biol. 6, 169. Borenfreund, E., Fitt, E. & Bendich, A. (1961). Nature, 191, 1375. Duggan, E. L. (1961). Biochem. Biophys. Res. Oomm. 6, 93. Emmanuel, C. F. (1960). Biochim. biophys. Acta, 42, 91, Felsenfeld, G., Sandeen, G. & von Hippel, P. H. (1963). Proc, Nat. Acad. Sei., Wash. 50, 644. Geiduschek, E. P. (1962). J. Mol. Biol. 4, 467. Langridge, R., Seeds, W. E., Wilson, H. R., Hooper, C. W., Wilkins, M. H. F. & Hamilton, L. D. (1957). J. Biophys. Biochem. Oytol. 3,767. Marmur, J. & Doty, P. (1961). J. Mol. Biol. 3, 585. Marmur, J. & Doty, P. (1962). J. Mol. Biol. 5, 109. Nash, D. & Plaut, W. (1964). Proc, Nat. Acad. Sci., Wash. 51, 731, Stahmann, M. A. & Bergmann, M. (1946). J. Organic Ohem. II, 586. Walker, P. M. B. (1957). Exp. Oell. Res. suppl, 4, 86. Walker, P. M. B., Leonard, J., Gibb, D. & Chamberlain, P. J. (1963). J. Sci. Instrum. 40, 166.