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Autoradiographic studies on the incorporation of amino acids into spermatozoa
Experimental
Cell
Research
17, 399-404
AUTORADIOGRAPHIC OF AMINO F. Laboratoire
(1959)
399
STUDIES ON THE INCORPORATION ACIDS INTO SPERMATOZOA MARTIN
de Morphologic
and animale,
Received
J. BRACHET Universit6
October
libre
de Bruxelles,
Belgique
21, 1958
IT is now generally assumed that ribonucleic acid (RNA) plays a direct role in protein synthesis 13, 61; for reviews, see [4, 51. Recent biochemical work by Hoagland et al. [lo] has contributed much to our knowledge in this field, in showing that both soluble and microsomal RNA are required for amino acid incorporation into proteins. There is, however, one apparent exception to the general rule that RNA is required for protein synthesis: it is the case of mature bull spermatozoa. ,4s shown by Hhargava [l 1, they incorporate amino acids into proteins to a very measurable extent; however, according to the studies of Rrachet [a], Friedlander and Fraser [9], Mauritzen et al. [12], ripe spermatozoa contain very little, if any, RNA. In order to solve this discrepancy, we have followed the incorporation of labelled amino acids into the proteins of mature spermatozoa, by two different methods: measurements with a Geiger-Miiller counter (as done by Bhargava [l]) and autoradiography. In order to gain more insight in the problem, additional autoradiographic studies on the incorporation of labelled amino acids into the proteins of unripe and ripe sperm cells have been undertaken, both in Virgo and in vitro.
MATERIAL
AND
TECHNIQUES
The main experiments were performed, as those of Bhargava [l], on mature bull spermatozoa:’ one hour after they had been collected, 1 ml of seminal fluid was added to 4.5 ml of Krebs medium containing in addition 5 per cent of fructose and 50 ~1 of an hydrolysate of radioactive Chlorella proteins (1.2 PC), obtained from Harwell. Incubation times varied from 30 min. to 4 hrs, at 37”, in the presenceof a O,jCO, (95/5) mixture. 0.2 ml aliquots were collected at that time and, after gentle centrifugation and 2 washingswith Krebs medium containing a caseinhydrolysate (in order to facilitate 1 We wish to express our sincere thanks from the Centre d’insCmination artificielle
to Mr. Dimitropoulos who kindly de Saint Servais-Namur.
provided
Experimental
Cell
the material
Research
17
F. Martin
and J. Rrachet
exchanges between labelled and unlabelled amino acids), the spermatozoa were fixed with methanol and smeared on microscope slides; they were then subjected to the autoradiography procedure described by Ficq [7] and left for exposition during 30 days; they were then stained with Unna’s methyl green-pyronine mixture. For the biochemical experiments, 0.5 ml aliquots were centrifuged for 5 min. at 1400 rpm.; the spermatozoa were washed twice with the casein hydrolysatc and precipitated with 30 per cent trichloracetic acid (150 ~1, 15 min.). Proteins were then extracted according to Simkin and Work [16] and their radioactivity, after drying, was measured with the counter; the protein content was estimated according to Lowry et al. [ll]. Frog testes were crushed in tap water and incubated, for 3 hours at 2O”C, with either 0.6 PC of radioactive Chlorella protein hydrolysate, or 0.2 PC of radioactive phenylalanine per ml. After two washings with either the casein hydrolysate or non-radioactive phenylalanine and centrifugation, the precipitate was fixed with methanol (20 min.), smeared and subjected to the autoradiography procedure as above. Rat mature sperm cells were collected from the epididymis and treated for 4 hours, as indicated above, with the following precursors: 2 &/ml phenylalanine; 2 &/ml glycine; 0.6 &/ml radioactive Chlorella protein hydrolysate. The autoradiography technique was the same as in the case of the bull spermatozoa. In other experiments, rat testes were crushed in Krebs medium containing 5 per cent fructose, incubated with the Chlorella protein hydrolysate and autoradiographed after an exposition of 48 days. The purpose of these experiments was to compare the radioactivity of the mature sperm cells to that of the sperm mother cells. Finally, in uiuo experiments were performed on male mice: they received intraperitoneally an injection of 20 ,& of the radioactive protein hydrolysate of Chlorella. The animals were sacrificed after 2 days; the testes were fixed with a 90 per cent alcohol, 10 per cent acetic acid mixture, and embedded in paraffin in the usual way. The sections were submitted to the autoradiography method of Ficq [7], the exposition time being 8 days.
EXPERIMENTAL
RESULTS
Ball spermatozoa.-The biochemical experiments on the specific radioactivity of the extracted proteins entirely confirm those of Rhargava [l 1, as indicated in Fig. 1, in which two independent experiments have been included. On the other hand, when the autoradiography method is used, it is found that, even after a 30-day exposition time, the radioactiaity of the sperm cells is the same as that of the backgroand. \Vhatever the incubation time with the radioactive precursor may be (varying from 1 to 4 hours), the autoradiograph counts give only 1 track per 8-13 sperm cells. A calculation of the surface of the sperm cells shows that the background alone would be of the order of 1 track per 9 sperm cells. Experimental
Cell
Research
17
Protein
synthesis
401
in spermatozoa
The origin of this discrepancy between the biochemical and autoradiographical results is easy to find: the autoradiographs show the presence of numerous, unidentifiable cell “debris”; their radioactivity is high (l-2 tracks per surface unit, as compared to 0.2 tracks per surface unit in the case of the background). In fact, the radioactivity of these contaminants (which might be of bacterial nature) can occasionally be so high as to preclude any accurate counting of the tracks.
Incubation
Fig.
time
1.
Frog testes.-Whatever the precursor used (phenylalanine or hydrolysate of radioactive Chlorellae), the sperm cells show again the same radioactivity as the background, after an l&day exposition, with the autoradiography method. On the other hand, unripe testis cells (spermatogonia, spermatocytes and spermatids) have, in the case of the Chlorella hydrolysate, a measurable radioactivity which increases almost linearly with time (1.2 tracks/cell after 30 min., 3 tracks after 3 hrs.; the background being substracted). Rat testes and mature rat spermatozoa collected from the epididymis.--Here again, whatever the precursor used and the exposition time of the autoradiographs, no activity exceeding that of the background can be detected in the ripe spermatozoa. Sperm mother cells, however, have a very measurable activity, the number of tracks per cell varying from 3.2 to 4.5, according to the length of the incubation with the radioactive precursor (30 min. to 2 hrs). Experimental
Cell Research
17
F. Marfin
402
and J. Brachef
In vivo experiments with mice.-\Xrhen the radioactive precursor (radioactive protein hydrolysate from Chlorella) is given to a male mouse, no radioactive spermatozoa are found in the epididymis after two days. All the cells of the testis are labelled and there is a very conspicuous and gradual decrease in the activity, from the periphery (spermatogonia) to the center (spermatozoa) of the tubes. There is a parallel decrease in the basophilia of the cells; basophilia is highest in the spcrmatogonia and lowest in the mature spermatozoa. It is difficult to decide, in sections of the testis, whether the spermatozoa are slightly labelled or not at all, because they are often intermingled with younger cells and because the lumen of the tubes shows, even in the absence of cells, a higher radioactivity than the background. As already found by Pelt [ 151, the RNA which is extruded from the cells during spermatogenesis shows very little radioactivity: these “residual bodies” are obviously very rich in RNA, but the latter must be in an abnormal condition, which does not enable it to fulfill its part in protein synthesis, The RNA of the “residual bodies” therefore behaves very much like the abnormal RNA which is synthesized when bacteria are treated with chloromycetin [13]. But, although the “residual bodies” show very little activity, the spermatozoa are still less active. DISCUSSION
In our experiments with bull spermatozoa, the radioactive material was studied simultaneously by the biochemical (counting) and autoradiography techniques. The biochemical method essentially confirmed Rhargava’s [ 1 ] results: the radioactivity of the proteins was of the order of 200-300 c.min./mg proteins after a 4 hr. incubation period. It is usually admitted that the sensitivity of the autoradiography technique is about 3 times higher than that of the counter (see Ficq [7], for a discussion): under conditions in which the counter registers 10 per cent of the radiation produced by the substances under study, autoradiography detects 30 per cent of the electrons produced by the cells. Let us assume that the mature sperm cells are entirely responsible for the radioactivity, as measured with the counter; we know, from Bhargava’s (1) own data that 1 mg of protein is extracted from approximately 4 X 107 cells. After 1 hr incubation with the radioactive precursor, the specific radioactivity of the proteins extracted from the spermatozoa is, approximately, 170 c.min./mg proteins. If the sensitivities of the two techniques (counting and autoradiography) were the same, the time of exposition required to find one track coming out Experimental
Cell
Research
17
Protein
synfhesis in spermatozoa
403
of every sperm cell Lvould be: 4 X 107/170 min., i.e. 139 days. Rut, if the sensitivity of the autoradiography technique is 3 times higher than that of the counting method, one should find one track per spermatozoijn after 46 days (the background being deduced). For 4 hours of incubation with the labelled precursor, one would expect to find as much as 4 tracks for 3 spermatozoa: that is, all the spermatozoa should be labelled, and some of them should even be twice labelled. However, the actual experiments show that, under these same experimental conditions, only one out of nine sperm cells is labelled: in fact, the spermatozoa are no more labelled than the background and most of the radioactivity is found in the cell “debris”. The conclusion, therefore, is that the mature spermatozoa show very little, if any, capacity for protein synthesis. Our experiments on rat and frog spermatozoa in vitro, as well as those on the mouse testis in uivo, entirely confirm this conclusion: the incorporation in the younger cells, which are rich in RNA, is much higher than in the ripe sperm cells. In fact, the radioactivity of the latter is negligible when the background is substracted. Our results on mouse testis entirely agree with the autoradiographic studies of Ficq and Brachrt [8] and of Niklas and Oehlert [14]: there is a close parallelism bctlvecn the basophilia (i.e. the RNA content of the cells) in all the organs and tissues which have been studied and the radioactivity due to amino acid incorporation into the proteins. \T’e must, therefore, conclude that mature spermatozoa make no exception to the general rule that RNA is involved in protein synthesis: they are very poor in RNA and, therefore, their protein anabolism is extremely lo\\-, if it exists at all.
SUMMARY
The incorporation of labelled amino acids into the proteins of mature spermatozoa and unripe sperm cells has been studied by counting and autoradiography methods: while, as shown by Rhargava [ 11, there is a measurable incorporation of labelled amino acids into the proteins in the case of bull semen, autoradiographic methods show that this incorporation occurs in unidentified cell “debris” and not in the spermatozoa themselves. The latter, lvhich contain very little or no RNA, have an extremely 101~protein anabolism, in accordance with the general rule that RNA is associated with protein synthesis. The incorporation of the labelled amino acids into the proteins of sperm mother cells is easily detected by autoradiography and is parallel to the basophilia of the cells. Experimental
Cell
Reserrrch
17
F. Marfin
and J. Brachef
REFERENCES P. M., Nature 179, 1120 (1957). J., Arch. Biol. 44, 519 (1933). 53, 207 (1942). J., in The Nucleic Acids, Vol. 2, p. 475. Ed. E. CHARGAFF and J. N. DAVIDSON, Academic Press, New York 1957. __ Biochemical Cytology. Academic Press, New York, 1957. CASPERSSON, T., Naturwissenschaffen 29, 33 (1941). FICQ, A., Arch. Biol. 65, 509 (1955). FICQ, A. and BRACHET, J., Expt/. Cell Research 11, 146 (1956). FRIEDLANDER, M. H. G. and FRASER, S. C., Expfl. Cell Research 3, 462 (1952). HOAGLAND, M. B., ZAMECNIK, P. C. and STEPHENSON, M. L., Biochim. et Biophys. Acfa24, 215 (1957). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J., J. biol. Chem. 193, 265 (1951). MAURITZEN, M., ROY, A. B. and STEDMAN, E., Proc. Roy. Sot. B 140, 18 (1957). NEIDHARDT, F. C. and GROS, F., Biochim. et Biophys. Acta 25, 513 (1957). NIKLAS, A. and OEHLERT, W., Beifr. pafhol. Anaf. u. allgem. Pafhol. 116, 92 (1956). PELC, S. R., Expfl. Ceil Research. 12, 320 (1957). SIMKIN, J. L. and WORK, T. S., Bioehem. J. 65, 307 (1957).
1. BHARGAVA,
2. BRACHET, 3. __ ibid. 4. BRACHET, 5. 6.
7. 8.
9. 10. 11. 12. 13. 14. 15. 16.
Experimental
Cell Research
17
Cell
Research
17, 399-404
AUTORADIOGRAPHIC OF AMINO F. Laboratoire
(1959)
399
STUDIES ON THE INCORPORATION ACIDS INTO SPERMATOZOA MARTIN
de Morphologic
and animale,
Received
J. BRACHET Universit6
October
libre
de Bruxelles,
Belgique
21, 1958
IT is now generally assumed that ribonucleic acid (RNA) plays a direct role in protein synthesis 13, 61; for reviews, see [4, 51. Recent biochemical work by Hoagland et al. [lo] has contributed much to our knowledge in this field, in showing that both soluble and microsomal RNA are required for amino acid incorporation into proteins. There is, however, one apparent exception to the general rule that RNA is required for protein synthesis: it is the case of mature bull spermatozoa. ,4s shown by Hhargava [l 1, they incorporate amino acids into proteins to a very measurable extent; however, according to the studies of Rrachet [a], Friedlander and Fraser [9], Mauritzen et al. [12], ripe spermatozoa contain very little, if any, RNA. In order to solve this discrepancy, we have followed the incorporation of labelled amino acids into the proteins of mature spermatozoa, by two different methods: measurements with a Geiger-Miiller counter (as done by Bhargava [l]) and autoradiography. In order to gain more insight in the problem, additional autoradiographic studies on the incorporation of labelled amino acids into the proteins of unripe and ripe sperm cells have been undertaken, both in Virgo and in vitro.
MATERIAL
AND
TECHNIQUES
The main experiments were performed, as those of Bhargava [l], on mature bull spermatozoa:’ one hour after they had been collected, 1 ml of seminal fluid was added to 4.5 ml of Krebs medium containing in addition 5 per cent of fructose and 50 ~1 of an hydrolysate of radioactive Chlorella proteins (1.2 PC), obtained from Harwell. Incubation times varied from 30 min. to 4 hrs, at 37”, in the presenceof a O,jCO, (95/5) mixture. 0.2 ml aliquots were collected at that time and, after gentle centrifugation and 2 washingswith Krebs medium containing a caseinhydrolysate (in order to facilitate 1 We wish to express our sincere thanks from the Centre d’insCmination artificielle
to Mr. Dimitropoulos who kindly de Saint Servais-Namur.
provided
Experimental
Cell
the material
Research
17
F. Martin
and J. Rrachet
exchanges between labelled and unlabelled amino acids), the spermatozoa were fixed with methanol and smeared on microscope slides; they were then subjected to the autoradiography procedure described by Ficq [7] and left for exposition during 30 days; they were then stained with Unna’s methyl green-pyronine mixture. For the biochemical experiments, 0.5 ml aliquots were centrifuged for 5 min. at 1400 rpm.; the spermatozoa were washed twice with the casein hydrolysatc and precipitated with 30 per cent trichloracetic acid (150 ~1, 15 min.). Proteins were then extracted according to Simkin and Work [16] and their radioactivity, after drying, was measured with the counter; the protein content was estimated according to Lowry et al. [ll]. Frog testes were crushed in tap water and incubated, for 3 hours at 2O”C, with either 0.6 PC of radioactive Chlorella protein hydrolysate, or 0.2 PC of radioactive phenylalanine per ml. After two washings with either the casein hydrolysate or non-radioactive phenylalanine and centrifugation, the precipitate was fixed with methanol (20 min.), smeared and subjected to the autoradiography procedure as above. Rat mature sperm cells were collected from the epididymis and treated for 4 hours, as indicated above, with the following precursors: 2 &/ml phenylalanine; 2 &/ml glycine; 0.6 &/ml radioactive Chlorella protein hydrolysate. The autoradiography technique was the same as in the case of the bull spermatozoa. In other experiments, rat testes were crushed in Krebs medium containing 5 per cent fructose, incubated with the Chlorella protein hydrolysate and autoradiographed after an exposition of 48 days. The purpose of these experiments was to compare the radioactivity of the mature sperm cells to that of the sperm mother cells. Finally, in uiuo experiments were performed on male mice: they received intraperitoneally an injection of 20 ,& of the radioactive protein hydrolysate of Chlorella. The animals were sacrificed after 2 days; the testes were fixed with a 90 per cent alcohol, 10 per cent acetic acid mixture, and embedded in paraffin in the usual way. The sections were submitted to the autoradiography method of Ficq [7], the exposition time being 8 days.
EXPERIMENTAL
RESULTS
Ball spermatozoa.-The biochemical experiments on the specific radioactivity of the extracted proteins entirely confirm those of Rhargava [l 1, as indicated in Fig. 1, in which two independent experiments have been included. On the other hand, when the autoradiography method is used, it is found that, even after a 30-day exposition time, the radioactiaity of the sperm cells is the same as that of the backgroand. \Vhatever the incubation time with the radioactive precursor may be (varying from 1 to 4 hours), the autoradiograph counts give only 1 track per 8-13 sperm cells. A calculation of the surface of the sperm cells shows that the background alone would be of the order of 1 track per 9 sperm cells. Experimental
Cell
Research
17
Protein
synthesis
401
in spermatozoa
The origin of this discrepancy between the biochemical and autoradiographical results is easy to find: the autoradiographs show the presence of numerous, unidentifiable cell “debris”; their radioactivity is high (l-2 tracks per surface unit, as compared to 0.2 tracks per surface unit in the case of the background). In fact, the radioactivity of these contaminants (which might be of bacterial nature) can occasionally be so high as to preclude any accurate counting of the tracks.
Incubation
Fig.
time
1.
Frog testes.-Whatever the precursor used (phenylalanine or hydrolysate of radioactive Chlorellae), the sperm cells show again the same radioactivity as the background, after an l&day exposition, with the autoradiography method. On the other hand, unripe testis cells (spermatogonia, spermatocytes and spermatids) have, in the case of the Chlorella hydrolysate, a measurable radioactivity which increases almost linearly with time (1.2 tracks/cell after 30 min., 3 tracks after 3 hrs.; the background being substracted). Rat testes and mature rat spermatozoa collected from the epididymis.--Here again, whatever the precursor used and the exposition time of the autoradiographs, no activity exceeding that of the background can be detected in the ripe spermatozoa. Sperm mother cells, however, have a very measurable activity, the number of tracks per cell varying from 3.2 to 4.5, according to the length of the incubation with the radioactive precursor (30 min. to 2 hrs). Experimental
Cell Research
17
F. Marfin
402
and J. Brachef
In vivo experiments with mice.-\Xrhen the radioactive precursor (radioactive protein hydrolysate from Chlorella) is given to a male mouse, no radioactive spermatozoa are found in the epididymis after two days. All the cells of the testis are labelled and there is a very conspicuous and gradual decrease in the activity, from the periphery (spermatogonia) to the center (spermatozoa) of the tubes. There is a parallel decrease in the basophilia of the cells; basophilia is highest in the spcrmatogonia and lowest in the mature spermatozoa. It is difficult to decide, in sections of the testis, whether the spermatozoa are slightly labelled or not at all, because they are often intermingled with younger cells and because the lumen of the tubes shows, even in the absence of cells, a higher radioactivity than the background. As already found by Pelt [ 151, the RNA which is extruded from the cells during spermatogenesis shows very little radioactivity: these “residual bodies” are obviously very rich in RNA, but the latter must be in an abnormal condition, which does not enable it to fulfill its part in protein synthesis, The RNA of the “residual bodies” therefore behaves very much like the abnormal RNA which is synthesized when bacteria are treated with chloromycetin [13]. But, although the “residual bodies” show very little activity, the spermatozoa are still less active. DISCUSSION
In our experiments with bull spermatozoa, the radioactive material was studied simultaneously by the biochemical (counting) and autoradiography techniques. The biochemical method essentially confirmed Rhargava’s [ 1 ] results: the radioactivity of the proteins was of the order of 200-300 c.min./mg proteins after a 4 hr. incubation period. It is usually admitted that the sensitivity of the autoradiography technique is about 3 times higher than that of the counter (see Ficq [7], for a discussion): under conditions in which the counter registers 10 per cent of the radiation produced by the substances under study, autoradiography detects 30 per cent of the electrons produced by the cells. Let us assume that the mature sperm cells are entirely responsible for the radioactivity, as measured with the counter; we know, from Bhargava’s (1) own data that 1 mg of protein is extracted from approximately 4 X 107 cells. After 1 hr incubation with the radioactive precursor, the specific radioactivity of the proteins extracted from the spermatozoa is, approximately, 170 c.min./mg proteins. If the sensitivities of the two techniques (counting and autoradiography) were the same, the time of exposition required to find one track coming out Experimental
Cell
Research
17
Protein
synfhesis in spermatozoa
403
of every sperm cell Lvould be: 4 X 107/170 min., i.e. 139 days. Rut, if the sensitivity of the autoradiography technique is 3 times higher than that of the counting method, one should find one track per spermatozoijn after 46 days (the background being deduced). For 4 hours of incubation with the labelled precursor, one would expect to find as much as 4 tracks for 3 spermatozoa: that is, all the spermatozoa should be labelled, and some of them should even be twice labelled. However, the actual experiments show that, under these same experimental conditions, only one out of nine sperm cells is labelled: in fact, the spermatozoa are no more labelled than the background and most of the radioactivity is found in the cell “debris”. The conclusion, therefore, is that the mature spermatozoa show very little, if any, capacity for protein synthesis. Our experiments on rat and frog spermatozoa in vitro, as well as those on the mouse testis in uivo, entirely confirm this conclusion: the incorporation in the younger cells, which are rich in RNA, is much higher than in the ripe sperm cells. In fact, the radioactivity of the latter is negligible when the background is substracted. Our results on mouse testis entirely agree with the autoradiographic studies of Ficq and Brachrt [8] and of Niklas and Oehlert [14]: there is a close parallelism bctlvecn the basophilia (i.e. the RNA content of the cells) in all the organs and tissues which have been studied and the radioactivity due to amino acid incorporation into the proteins. \T’e must, therefore, conclude that mature spermatozoa make no exception to the general rule that RNA is involved in protein synthesis: they are very poor in RNA and, therefore, their protein anabolism is extremely lo\\-, if it exists at all.
SUMMARY
The incorporation of labelled amino acids into the proteins of mature spermatozoa and unripe sperm cells has been studied by counting and autoradiography methods: while, as shown by Rhargava [ 11, there is a measurable incorporation of labelled amino acids into the proteins in the case of bull semen, autoradiographic methods show that this incorporation occurs in unidentified cell “debris” and not in the spermatozoa themselves. The latter, lvhich contain very little or no RNA, have an extremely 101~protein anabolism, in accordance with the general rule that RNA is associated with protein synthesis. The incorporation of the labelled amino acids into the proteins of sperm mother cells is easily detected by autoradiography and is parallel to the basophilia of the cells. Experimental
Cell
Reserrrch
17
F. Marfin
and J. Brachef
REFERENCES P. M., Nature 179, 1120 (1957). J., Arch. Biol. 44, 519 (1933). 53, 207 (1942). J., in The Nucleic Acids, Vol. 2, p. 475. Ed. E. CHARGAFF and J. N. DAVIDSON, Academic Press, New York 1957. __ Biochemical Cytology. Academic Press, New York, 1957. CASPERSSON, T., Naturwissenschaffen 29, 33 (1941). FICQ, A., Arch. Biol. 65, 509 (1955). FICQ, A. and BRACHET, J., Expt/. Cell Research 11, 146 (1956). FRIEDLANDER, M. H. G. and FRASER, S. C., Expfl. Cell Research 3, 462 (1952). HOAGLAND, M. B., ZAMECNIK, P. C. and STEPHENSON, M. L., Biochim. et Biophys. Acfa24, 215 (1957). LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J., J. biol. Chem. 193, 265 (1951). MAURITZEN, M., ROY, A. B. and STEDMAN, E., Proc. Roy. Sot. B 140, 18 (1957). NEIDHARDT, F. C. and GROS, F., Biochim. et Biophys. Acta 25, 513 (1957). NIKLAS, A. and OEHLERT, W., Beifr. pafhol. Anaf. u. allgem. Pafhol. 116, 92 (1956). PELC, S. R., Expfl. Ceil Research. 12, 320 (1957). SIMKIN, J. L. and WORK, T. S., Bioehem. J. 65, 307 (1957).
1. BHARGAVA,
2. BRACHET, 3. __ ibid. 4. BRACHET, 5. 6.
7. 8.
9. 10. 11. 12. 13. 14. 15. 16.
Experimental
Cell Research
17