- Email: [email protected]
Fluorescence analysis of late DNA replication in mouse metaphase chromosomes using BUdR and 33258 Hoechst
438
Preliminary notes
enhanced, and numerous procentrioles and centrioles are present in many pituicytes. The cilia are mainly of the 9+0 type, although a few typical 9+2 cilia have been observed. In the presence of VCR, in addition to numerous *large paracrystals of tubulin [4] and apart from centrioles and occasional cilia, macrotubules are frequently observed (fig. 2). They have a diameter of 48 nm, are often closely related to normal microtubules and are frequently found in axonal endings close to the cell membrane. They present a helical structure resembling some of the double (or “decorated”) microtubules described by Behnke [S]. These experiments, confirming our previous findings in vivo [l, 2, 31, indicate that newly formed centrioles can appear in great numbers within a short time in vitro and lead to cilia growth. This takes place already in saline solutions, and is paradoxically enhanced by the microtubule poisons colchicine and VCR. The formation of centrioles from dense bodies is closely similar to that described in other tissues [6]. A few other observations indicate that colchicine does not necessarily prevent the formation of centrioles and cilia [7, 81. It has been observed that in some conditions it could aggregate tubulin [9]. The apparent influence of Mg*+ ions in the saline solutions is in agreement with the known role of Mg*+ in tubulin polymerization in vitro [IO]. Our experiments appear to confirm the hypothesis presented in 1975 [2] that the tubulin association in the form of centrioles and cilia, which are structures resistant to microtubule poisoning, is increased by colchicine and VCR. Weisenberg & Rosenfeld [Ill, observing the formation of microtubule organizing centers (MTOC) in eggs treated with colchicine, also suggest that these structures may be Exp Cell Res 99 (1976)
composed of tubulin in a state insensitive to colchicine. The role of changes of the ionic milieu on pituicytes, the function of which is to control the secretion of antidiuretic hormone, will be the subject of further work. The authors wish to thank Mrs A. M. HunninckCouck for her devoted and skillful technical assistance and Mrs D. Libert-Kaca for the careful typing of the manuscript. They are endebted to the fiim EliLilly for providing samples of Oncovin. This work was supported by a grant from the Belgian National Fund for Scientific Research and by grant No. 1120 from the Belgian National Fund for Medical Research.
References 1. Hubert, J P, Flament-Durand, J & Dustin, P, Cell tissue res 149 (1974) 349. 2. Dustin, P, Hubert, J P & Flament-Durand, J, Ann NY acad sci 253 (1975) 670. 3. Dustin, P, Hubert, J P & Flament-Durand, J, In Microtubules and microtubule inhibitors (ed M Borgers & M de Brabander). North Holland, Amsterdam (1975). 4. Rufener, C, Rouiller, C & Orci, L, Experientia 28 (1972) 837. 5. Behnke. 0. Nature 57 (1975) 709. 6. Dirksen, E’R, J cell bid1 41 (1971) 286. 7. Stubblefield, E & Brinkley, B R, J cell biol 30 (1966) 645. 8. Wunderlich, F Jr Heumann, H G, Cytobiologie 10 (1974) 140. 9. Weisenberg, R C & Timasheff, S N, Biochemistry 9 (1970) 4110. 10. Borisy, G G, Marcum, J M, Olmsted, J B, Murphy, D B & Johnson, K A, Ann NY acad sci 253 (1975) 107. 11. Weisenberg, R C & Rosenfeld, A, J cell biol 64 (1975) 42. Received December 11, 1975 Accepted January 9, 1976
Fluorescence analysis of late DNA replication in mouse metaphase chromosomes using BUdR and 33258 Hoechst K. MADAN,’ J. W. ALLEN,* P. S. GERALD* and S. A. LATTZ, Winical Genetics Division, Children’s Hospital Medical Center, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA, and Cytogenetics Laboratory, Department of Obstetrics and ‘Gynecology, Free University, Amsterdam, The Netherlands Summary. A late replicating X or Y chromosome can be detected by 33258 Hoechst staining and fluorescence microscopy in a large proportion of female or
Preliminary notes male mouse embryo cells, respectively, which have been cultured in medium containing Sbromodeoxvuridine (BUdR) for part of one DNAsynthesis periok The observed distribution of late renlicatine chromosome regions also includes centromeric heterochromatin and some quinacrine positive bands.
Differentiation between female X chromosomes based on DNA replication kinetics has proved more difficult in the mouse than in man. Autoradiographic studies using [3H]TdR administered to cells late in the DNA synthesis (S) period allow unambiguous identification of a late replicating X in only a small fraction of the cells from female mice [ 1, 21. Detection of X chromosome replication asynchrony in female mice is somewhat more successful if cells are labelled early in the S period and differentiation is based on the delay in initiation of DNA synthesis rather than on the delay in its completion [4]. The mouse Y, unlike the human Y [5], is one of the latest replicating chromosomes [2, 3,4], and its identification by autoradiography has proved less difflcult. In contrast to the situation in human cells [6, 71, both quinacrine and Giemsa techniques appear capable of distinguishing between the two X chromosomes of female mice [8, 91, although the results contain no information about the internal order of replication. The autoradiographic techniques referred to above possess a resolution which is inherently limited by spatial and statistical factors. More definitive evaluation of the replication characteristics of the mouse X and Y, as well as the autosomes, is now possible with newly-developed optical methods for detecting DNA synthesis. Under appropriate conditions, substitution of 5-bromodeoxyuridine (BUdR) for thymidine (TdR) can be detected in metaphase chromosomes by quenching of 33258 Hoechst [ 10, 11, 121or acridine orange fluorescence [ 13, 14, 151,by pale Giemsa staining [16, 17,
439
181,or by reaction with BUdR-specific antibodies [ 191.This approach has already been utilized to investigate the replication kinetics of human chromosomes [20] as well as to detect sister chromatid exchanges [2 1, 221 and regions containing DNA with an asymmetric distribution of TdR between complementary strands [23, 24, 2.51. In the present report, BUdR-Hoechst techniques are utilized to study replication kinetics in primary cultures of embryonic mouse tissue. If BUdR is incorporated into cells late in the S period, late replicating chromosome regions exhibit dull 33258 Hoechst fluorescence. Conversely, if cells are grown in BUdR-containing medium for most of one replication cycle and then exposed to a terminal preharvest pulse of TdR, late replicating regions exhibit bright 33258 Hoechst fluorescence against a BUdR-suppressed background. These techniques are highly effective at identifying the late replicating female X and male Y chromosomes in mice, and further provide information about the internal sequence of chromosome replication. Materials and Methods Individual or multiple embryos (13-15 day) from CD-1 mice which were purchased from Charles River Breeding Labs were minced and trypsinized, and the tissue was cultured in Ham’s F-10 medium containing 10% fetal calf serum. On the third day following initiation of culture, the cells were transferred to 150 mm Petri dishes (approx. lo6 cells/dish). Approx. 34 h after the start of the subcultures, Colcemid (0.1 @g/ml) was added, and 2 h later the cells were harvested. Prior to harvest, each culture was given one of the following treatments: (a) Sb~omodeoxyuridine (BUdR) and deoxycytidine were added (final cont. of each 10m4M) to the cultures either 6 or 9 h before harvesting (B-pulse protocol). Deoxycytidine was added to red&e ioxic dffects of BUdR [26]. (b) BUdR and deoxycytidine (concentrations as above) were added 18 h before harvesting. The medium was changed, and thymidine was added (final cont. 1.2~ 10eJM) 31, 6, or 9 h before harvesting (T pulse protocol). The slides were made by the conventional air-drying technique and were stained with 33258 Hoechst‘ [27, 281 as described previously [ll, 12, 131.In some Exp Cell Ret 99 (1976)
440
Preliminary
notes
Fig. 1. Fluorescence detection of the late replicating mouse Y chromosome. Cultured mouse embryonic cells were exposed to BUdR for 12 h (a, b) or 9 h (c, d) followed by growth in medium containing deoxythymidine but not BUdR for 6 h (a, b) or 9 h (c, 6). One
cell was stained with quinacrine for chromosome identification (a), destained, and restained with 33258 Hoechst (b). c and d were stained directly with 33258 Hoechst. The Y chromosome is evident by unusually bright (b, c) or dull (d) fluorescence (arrows).
cases, slides were first stained with quinacrine dihydrochloride for chromosome identification, destained after photography [22], and then restained with Hoechst 33258.
male cells), a single small asynchronously replicating chromosome, identified as the Y by quinacrine staining and by the absence of a bright centromere following staining with 33258 Hoechst [27] was apparent (fg 1). In the second type (presumptively female), a single large asynchronous chromo-
Results and Discussion
Two types of BUdR-incorporating cells were observed. In the first (presumptively Exp Cell Res 99 (1976)
Preliminary
notes
44 I
Fig.
2. Fluorescence detection of the late replicating mouse X chromosomes. Cultured mouse embryo cells were exposed to BUdR for 9 h followed bv prowth in medium containing deoxyrhymidine for 9 h. Slides
were stained with 33258 Hoechst. The late rephcating mouse X chromosome is indicated by an arrow. Late replication is associated with bright fluorescence in u. 6, and c but by dull fluorescence in d.
some was observed (fig. 2). The fluorescence pattern of the latter chromosome resembles the quinacrine banding pattern of the mouse X [29, 301. Cultures prepared from single embryos exhibited either one cell type or the other, while cultures contaming a mixture of tissue from several
male and female embryos exhibit both cell types. While complementary 33258 Hoechst fluorescence patterns were obtained with the two growth protocols, more distinctive patterns were apparent with the T-pulse protocol, in which BUdR incorporation Exp Cell Rex YY i/Y761
442
Preliminary
notes
Fig. 3. Examples of 33258 Hoechst fluorescence patterns ilhrstrating terminal replication in female mouse X chromosomes. Cells were grown according to the T pulse protocol as described in the caption to fig. 1. Pairs of chromosomes in (a) were sequentially stained with quinacrine (top row) and then 33258 Hoechst
(bottom row). All pairs of chromosomes in (b) were stained with 33258 Hoechst. In each r&r, the nresumptive late replicating X (as judged by brighter 33258 Hoechst fluorescence) is positioned to the right of its homologue.
from the start of S was followed by a preharvest pulse of deoxythymidine. Out of 254 male cells examined, 191 (75 %) exhibited a late replicating Y chromosome with strikingly bright fluorescence (fig. 1b, c). In another 17 cells (7 %), the Y chromosome fluoresced much less brightly than did the other chromosomes (fig. Id). These last cells presumably completed replication during the BUdR administration, incorporated virtually none of the TdR pulse, and were trapped at the subsequent metaphase. In 4 cells (2%), bright fluorescence was observed over a few autosome arms but not over the Y. In the remaining 42 male cells, fluorescence banding was not observed. In these, overall fluorescence was dull, and the centromeres exhibited either the lateral asymmetry expected [23] after one cycle of BUdR incorporation, or entire centromeres fluoresced brightly. Out of 298 female cells examined, 186 (62%) exhibited a single brightly fluorescent chromosome with the banding pattern
expected for a mouse X (fig. 2a-c). In 28 cells (9%), a single X chromosome displayed strikingly dull fluorescence (fig. 2d). In 16 cells (5%) bright fluorescence was present in the autosome arms without there being evidence of a late replicating X. The remaining 68 cells exhibited either uniform bright fluorescence, two brightly fluorescent X chromosomes, or generalized dull fluorescence with the possible exception of bright centromeres. A small number of cells from cultures treated with the converse (B-pulse) protocol was analysed. A late replicating X or Y could be distinguished by its dull fluorescence in virtually all of those cells incorporating BUdR (as judged by quenching of 33258 Hoechst fluorescence in any chromosome region). The above success rates for identifying the late replicating X or Y chromosomes compare favorably with those reported by previous autoradiogranhic analysis [ 141. Fluorescence analysis of late replication
Exp Cell Res 99 (1976)
Preliminary notes
Fig. 4. Examples of 33258 Hoechst fluorescence patterns illustrating terminal replication in mouse Y chromosomes. Cells were grown in medium containing BUdR except for a 3f h terminal pulse of deoxycytidine (A, B, C) or in control medium except for a 6-h terminal pulse of BUdR (D). Chromosome (A) exhibits dull fluorescence, presumably because a negligible amount of the deoxythymidine pulse had been incorporated. Chromosomes B and C exhibit slightly brighter fluorescence, more marked in the distal half of the chromosome. Chromosome D shows a complementary pattern, with dull fluorescence accentuated in the distal half of the chromosome. Note the lack of selective bright fluorescence in the centromerit regions consistent with the reported absence of mouse satellite DNA from the Y chromosome [33].
also permits additional differentiation within and between mouse chromosomes. In cells administered a 3f or a 6-h terminal pulse of TdR, bright fluorescence was usually localized to the centromeric regions and the late replicating X or Y chromosome (e.g. fig. 1b). Identification of deoxythymidine incorporation in centromeric areas was relatively unambiguous, since a single cycle of BUdR incorporation into these regions results in a lateral asymmetry of 33258 Hoechst fluorescence [23]. Bright fluorescence of an X or a Y was not observed in the absence of bright centromeres. However, in a few cells, a variable proportion of the centromeres exhibited bilaterally bright fluorescence, and chromosome arm fluorescence was uniformly dull. These results are consistent with previous reports [31, 321 that mouse satellite DNA, which is located in the centromeric regions [33], replicates late in S. They suggest that the
443
timing of satellite DNA replication may not be the same in all chromosomes. Hsu & Markvong [34] have similarly observed that very late replication in Mus muscufus, as determined by autoradiography, did not involve all centromere regions, even when grains were present over some of the chromosome arms. These authors obtained data with both autoradiography and a modified BUdR-Giemsa technique [ 171 indicating that late replication followed a banded distribution in the chromosome arms of Mus dunni. Similarly, we observed that in many cells administered a terminal deoxythymidine pulse (e.g. fig. 2c, 9-h pulse), extensive bright fluorescence occurred in a banded distribution in the autosome arms. This fluorescence pattern consists of a subset of the quinacrine bands of mouse chromosomes [29, 301, together with the C-band positive centromeres. Within the X, late replication is most pronounced in and around the centromeric region, and in a prominent band in the distal region of the chromosome arm (fig. 3). Unlike the results with human female lymphocytes [35, 361, qualitative differences in the internal order of replication between the two mouse X chromosomes have not thus far been observed. Late replication in the mouse Y usually appears to involve the entire chromosome. In an occasional cell, late replication is somewhat more pronounced in a localized region just distal to the middle of this chromosome (fig. 4). The technique of BUdR incorporation and staining with Hoechst 33258 for detecting late DNA replicating regions of chromosomes appears to be useful for detecting asynchronous replication of X and Y chromosomes in mice. Because of the high percentage of female cells showing a late replicating X and the sharp demarcation of the early and late replicating re-
444
Preliminary notes
gions, this technique should prove useful in examining X-autosome translocations [37] for possible position-dependent alterations in replication kinetics [38]. In addition, the method may be especially useful for detecting small fragments of late-replicating X or Y chromatin translocated to autosomal chromosomes [39]. This work was supported by a grant from the National Foundation March of Dimes (l-353) and a research career development award (GM 00122) from the National Institute of Genera1 Medical Sciences.
30. Miller, D A & Miller, 0 J. Science 178 f1972) 949. 31. Tobia, A, Schildkraut, C L & Maio, J J,‘J mdl biol 54 (1970) 499. 32. - Biochim biophys acta 246 (1971) 258. 33. Pardue, M L & Gall, J, Science 168(1970) 1356. 34. Hsu, T C & Markvong, A, Chromosoma 5 1 (1975)
_111.__.
35. Willard, H & Latt, S, Am j human genet (1976). In press. 36. Latt, S A, Exp cell res 86 (1974) 412. 37. y?, S & Cattanach, B M, Cytogenetics 1 (1%2) 38. Either, E, Adv genet 15 (1970) 175. 39. Cattanach, B M, Birth defects (ed A Motulsky & F Ebling) p. 129. Excerpta Medica, Amsterdam (1974). Received December 17, 1975 Accepted January 21, 1976
References Evans, H J, Ford, C E, Lyon, M F & Gray, J, Nature 206 (1965) 900. 2. Galton, M & Holt, S F, Exp cell res 37 (1965) 111. 3. Tiepolo, I, Fraccaro, M, Hulten, M, Lindsten, J, Mannini, A & Ming, P M L, Cytogenetics 6 (1%7) 51. 4. Nesbitt, M N & Gartler, S M, Cytogenetics 9 (1970) 212. 5. Craig, A P & Shaw, M W, Chromosoma 32 (1971) 364. 6. Caspersson, T, Lindsten, J, Lomakka, G, Msller, A & Zech, L, Int rev exp path01 11 (1973) 1. 7. Hsu, T C, Ann rev genet 7 (1974) 153. Kanda, N, Exp cell res 80 (1973) 463. ;: Takagi, N, Exp cell res 86 (1974) 127. 10. Latt, S A, Proc natl acad sci US 70 (1973) 3395. 11. - J histochem cytochem 22 (1974) 478. 12. Latt, S A, Juergens, L A, Stetten, G, Willard, H F & Scher, C D, J histochem cytochem 23 (1975) 493. 13. DutrilIaux, M B, Laurent, C, Couturier, J & LeJeune, J, Compt rend acad sci D 276 (1973) 3 175. 14. Kato, H, Nature 251 (1974) 70. 15. Takagi, N & Sasaki, M, Nature 256 (1975) 640. 16. Perry, P&Wolff, S, Nature 251 (1974) 156. 17. Korenberg, J & Freedlender, E, Chromosoma 48 (1974) 355. 18. Kim, M A, Humangenetik 25 (1974) 179. 19. Gratzner, H, Ingram, D, Leif, R D & Castro, A, Exp cell res 95 (1975) 88. 20. Latt, S A, Somatic cell genet 1 (1975) 293. 21. - Science 185 (1974) 74. 22. - Proc natl acad sci US 71 (1974) 3162. 23. Lin, M S, Latt, S A & Davidson, R L, Exp cell res 86 (1974) 392. 24. Latt, S A, Davidson, R L, Lin, M S & Gerald, P S, Exp cell res 87 (1974) 425. 2s. Angell, R & Jacobs, P A, Chromosoma 51 (1975) 301. 26. Meuth, M & Green, H, Cell 2 (1974) 109. 27. Hilwig, I & Gropp, A, Exp cell res 75 (1972) 122. 28. Loewe, H & Urbanietz, J, Arzneimittelforsch 24 (1974) 1927. 29. Francke, U & Nesbitt, M, Proc natl acad sci US 68 (1971) 2918. 1.
Exp Cell Res 99 (I 976)
Separation of Kupffer and endothelial cells of the rat liver by centrifugal elutriation D. L. KNOOK and E. CH. SLEYSTER, Znsriturefor Experimental lands
Gerontology
TNO, Rijswijk,
The Nether-
Isolated non-parenchymal cells from rat liver were separated by centrifugal elutriation into two fractions consisting of structurally intact Kupffer and endothelial cells with purities of 91 and 95 %, respectively. Purified Kupffer and endothelial cells showed nearly equal specific activities for the lysosomal enzyme acid phosphatase, whereas the specific activity of cathepsin D was about 3 times higher in Kupffer cells. It was calculated that a significant amount of the cathepsin D activity in the liver is present in the Kupffer cells.
Summary.
The two main cell types in the mammalian liver are parenchymal cells and sinusoidal lining cells. Although the latter cell group occupies only 3 % of the total liver volume in the young adult rat, at least one-third of all liver cells are sinusoidal cells [l]. The non-parenchymal cells in the sinusoidal area have usually been described as Kupffer cells. Electron microscopic observations, however, showed, in addition to Kupffer cells, the presence of a number of cell types such as endothelial cells, fat-storing cells, pit cells, monocytes and other white blood cells [2]. In view of their number and function, Kupffer and endothelial cells are by far the most important sinusoidal lining
Preliminary notes
enhanced, and numerous procentrioles and centrioles are present in many pituicytes. The cilia are mainly of the 9+0 type, although a few typical 9+2 cilia have been observed. In the presence of VCR, in addition to numerous *large paracrystals of tubulin [4] and apart from centrioles and occasional cilia, macrotubules are frequently observed (fig. 2). They have a diameter of 48 nm, are often closely related to normal microtubules and are frequently found in axonal endings close to the cell membrane. They present a helical structure resembling some of the double (or “decorated”) microtubules described by Behnke [S]. These experiments, confirming our previous findings in vivo [l, 2, 31, indicate that newly formed centrioles can appear in great numbers within a short time in vitro and lead to cilia growth. This takes place already in saline solutions, and is paradoxically enhanced by the microtubule poisons colchicine and VCR. The formation of centrioles from dense bodies is closely similar to that described in other tissues [6]. A few other observations indicate that colchicine does not necessarily prevent the formation of centrioles and cilia [7, 81. It has been observed that in some conditions it could aggregate tubulin [9]. The apparent influence of Mg*+ ions in the saline solutions is in agreement with the known role of Mg*+ in tubulin polymerization in vitro [IO]. Our experiments appear to confirm the hypothesis presented in 1975 [2] that the tubulin association in the form of centrioles and cilia, which are structures resistant to microtubule poisoning, is increased by colchicine and VCR. Weisenberg & Rosenfeld [Ill, observing the formation of microtubule organizing centers (MTOC) in eggs treated with colchicine, also suggest that these structures may be Exp Cell Res 99 (1976)
composed of tubulin in a state insensitive to colchicine. The role of changes of the ionic milieu on pituicytes, the function of which is to control the secretion of antidiuretic hormone, will be the subject of further work. The authors wish to thank Mrs A. M. HunninckCouck for her devoted and skillful technical assistance and Mrs D. Libert-Kaca for the careful typing of the manuscript. They are endebted to the fiim EliLilly for providing samples of Oncovin. This work was supported by a grant from the Belgian National Fund for Scientific Research and by grant No. 1120 from the Belgian National Fund for Medical Research.
References 1. Hubert, J P, Flament-Durand, J & Dustin, P, Cell tissue res 149 (1974) 349. 2. Dustin, P, Hubert, J P & Flament-Durand, J, Ann NY acad sci 253 (1975) 670. 3. Dustin, P, Hubert, J P & Flament-Durand, J, In Microtubules and microtubule inhibitors (ed M Borgers & M de Brabander). North Holland, Amsterdam (1975). 4. Rufener, C, Rouiller, C & Orci, L, Experientia 28 (1972) 837. 5. Behnke. 0. Nature 57 (1975) 709. 6. Dirksen, E’R, J cell bid1 41 (1971) 286. 7. Stubblefield, E & Brinkley, B R, J cell biol 30 (1966) 645. 8. Wunderlich, F Jr Heumann, H G, Cytobiologie 10 (1974) 140. 9. Weisenberg, R C & Timasheff, S N, Biochemistry 9 (1970) 4110. 10. Borisy, G G, Marcum, J M, Olmsted, J B, Murphy, D B & Johnson, K A, Ann NY acad sci 253 (1975) 107. 11. Weisenberg, R C & Rosenfeld, A, J cell biol 64 (1975) 42. Received December 11, 1975 Accepted January 9, 1976
Fluorescence analysis of late DNA replication in mouse metaphase chromosomes using BUdR and 33258 Hoechst K. MADAN,’ J. W. ALLEN,* P. S. GERALD* and S. A. LATTZ, Winical Genetics Division, Children’s Hospital Medical Center, Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA, and Cytogenetics Laboratory, Department of Obstetrics and ‘Gynecology, Free University, Amsterdam, The Netherlands Summary. A late replicating X or Y chromosome can be detected by 33258 Hoechst staining and fluorescence microscopy in a large proportion of female or
Preliminary notes male mouse embryo cells, respectively, which have been cultured in medium containing Sbromodeoxvuridine (BUdR) for part of one DNAsynthesis periok The observed distribution of late renlicatine chromosome regions also includes centromeric heterochromatin and some quinacrine positive bands.
Differentiation between female X chromosomes based on DNA replication kinetics has proved more difficult in the mouse than in man. Autoradiographic studies using [3H]TdR administered to cells late in the DNA synthesis (S) period allow unambiguous identification of a late replicating X in only a small fraction of the cells from female mice [ 1, 21. Detection of X chromosome replication asynchrony in female mice is somewhat more successful if cells are labelled early in the S period and differentiation is based on the delay in initiation of DNA synthesis rather than on the delay in its completion [4]. The mouse Y, unlike the human Y [5], is one of the latest replicating chromosomes [2, 3,4], and its identification by autoradiography has proved less difflcult. In contrast to the situation in human cells [6, 71, both quinacrine and Giemsa techniques appear capable of distinguishing between the two X chromosomes of female mice [8, 91, although the results contain no information about the internal order of replication. The autoradiographic techniques referred to above possess a resolution which is inherently limited by spatial and statistical factors. More definitive evaluation of the replication characteristics of the mouse X and Y, as well as the autosomes, is now possible with newly-developed optical methods for detecting DNA synthesis. Under appropriate conditions, substitution of 5-bromodeoxyuridine (BUdR) for thymidine (TdR) can be detected in metaphase chromosomes by quenching of 33258 Hoechst [ 10, 11, 121or acridine orange fluorescence [ 13, 14, 151,by pale Giemsa staining [16, 17,
439
181,or by reaction with BUdR-specific antibodies [ 191.This approach has already been utilized to investigate the replication kinetics of human chromosomes [20] as well as to detect sister chromatid exchanges [2 1, 221 and regions containing DNA with an asymmetric distribution of TdR between complementary strands [23, 24, 2.51. In the present report, BUdR-Hoechst techniques are utilized to study replication kinetics in primary cultures of embryonic mouse tissue. If BUdR is incorporated into cells late in the S period, late replicating chromosome regions exhibit dull 33258 Hoechst fluorescence. Conversely, if cells are grown in BUdR-containing medium for most of one replication cycle and then exposed to a terminal preharvest pulse of TdR, late replicating regions exhibit bright 33258 Hoechst fluorescence against a BUdR-suppressed background. These techniques are highly effective at identifying the late replicating female X and male Y chromosomes in mice, and further provide information about the internal sequence of chromosome replication. Materials and Methods Individual or multiple embryos (13-15 day) from CD-1 mice which were purchased from Charles River Breeding Labs were minced and trypsinized, and the tissue was cultured in Ham’s F-10 medium containing 10% fetal calf serum. On the third day following initiation of culture, the cells were transferred to 150 mm Petri dishes (approx. lo6 cells/dish). Approx. 34 h after the start of the subcultures, Colcemid (0.1 @g/ml) was added, and 2 h later the cells were harvested. Prior to harvest, each culture was given one of the following treatments: (a) Sb~omodeoxyuridine (BUdR) and deoxycytidine were added (final cont. of each 10m4M) to the cultures either 6 or 9 h before harvesting (B-pulse protocol). Deoxycytidine was added to red&e ioxic dffects of BUdR [26]. (b) BUdR and deoxycytidine (concentrations as above) were added 18 h before harvesting. The medium was changed, and thymidine was added (final cont. 1.2~ 10eJM) 31, 6, or 9 h before harvesting (T pulse protocol). The slides were made by the conventional air-drying technique and were stained with 33258 Hoechst‘ [27, 281 as described previously [ll, 12, 131.In some Exp Cell Ret 99 (1976)
440
Preliminary
notes
Fig. 1. Fluorescence detection of the late replicating mouse Y chromosome. Cultured mouse embryonic cells were exposed to BUdR for 12 h (a, b) or 9 h (c, d) followed by growth in medium containing deoxythymidine but not BUdR for 6 h (a, b) or 9 h (c, 6). One
cell was stained with quinacrine for chromosome identification (a), destained, and restained with 33258 Hoechst (b). c and d were stained directly with 33258 Hoechst. The Y chromosome is evident by unusually bright (b, c) or dull (d) fluorescence (arrows).
cases, slides were first stained with quinacrine dihydrochloride for chromosome identification, destained after photography [22], and then restained with Hoechst 33258.
male cells), a single small asynchronously replicating chromosome, identified as the Y by quinacrine staining and by the absence of a bright centromere following staining with 33258 Hoechst [27] was apparent (fg 1). In the second type (presumptively female), a single large asynchronous chromo-
Results and Discussion
Two types of BUdR-incorporating cells were observed. In the first (presumptively Exp Cell Res 99 (1976)
Preliminary
notes
44 I
Fig.
2. Fluorescence detection of the late replicating mouse X chromosomes. Cultured mouse embryo cells were exposed to BUdR for 9 h followed bv prowth in medium containing deoxyrhymidine for 9 h. Slides
were stained with 33258 Hoechst. The late rephcating mouse X chromosome is indicated by an arrow. Late replication is associated with bright fluorescence in u. 6, and c but by dull fluorescence in d.
some was observed (fig. 2). The fluorescence pattern of the latter chromosome resembles the quinacrine banding pattern of the mouse X [29, 301. Cultures prepared from single embryos exhibited either one cell type or the other, while cultures contaming a mixture of tissue from several
male and female embryos exhibit both cell types. While complementary 33258 Hoechst fluorescence patterns were obtained with the two growth protocols, more distinctive patterns were apparent with the T-pulse protocol, in which BUdR incorporation Exp Cell Rex YY i/Y761
442
Preliminary
notes
Fig. 3. Examples of 33258 Hoechst fluorescence patterns ilhrstrating terminal replication in female mouse X chromosomes. Cells were grown according to the T pulse protocol as described in the caption to fig. 1. Pairs of chromosomes in (a) were sequentially stained with quinacrine (top row) and then 33258 Hoechst
(bottom row). All pairs of chromosomes in (b) were stained with 33258 Hoechst. In each r&r, the nresumptive late replicating X (as judged by brighter 33258 Hoechst fluorescence) is positioned to the right of its homologue.
from the start of S was followed by a preharvest pulse of deoxythymidine. Out of 254 male cells examined, 191 (75 %) exhibited a late replicating Y chromosome with strikingly bright fluorescence (fig. 1b, c). In another 17 cells (7 %), the Y chromosome fluoresced much less brightly than did the other chromosomes (fig. Id). These last cells presumably completed replication during the BUdR administration, incorporated virtually none of the TdR pulse, and were trapped at the subsequent metaphase. In 4 cells (2%), bright fluorescence was observed over a few autosome arms but not over the Y. In the remaining 42 male cells, fluorescence banding was not observed. In these, overall fluorescence was dull, and the centromeres exhibited either the lateral asymmetry expected [23] after one cycle of BUdR incorporation, or entire centromeres fluoresced brightly. Out of 298 female cells examined, 186 (62%) exhibited a single brightly fluorescent chromosome with the banding pattern
expected for a mouse X (fig. 2a-c). In 28 cells (9%), a single X chromosome displayed strikingly dull fluorescence (fig. 2d). In 16 cells (5%) bright fluorescence was present in the autosome arms without there being evidence of a late replicating X. The remaining 68 cells exhibited either uniform bright fluorescence, two brightly fluorescent X chromosomes, or generalized dull fluorescence with the possible exception of bright centromeres. A small number of cells from cultures treated with the converse (B-pulse) protocol was analysed. A late replicating X or Y could be distinguished by its dull fluorescence in virtually all of those cells incorporating BUdR (as judged by quenching of 33258 Hoechst fluorescence in any chromosome region). The above success rates for identifying the late replicating X or Y chromosomes compare favorably with those reported by previous autoradiogranhic analysis [ 141. Fluorescence analysis of late replication
Exp Cell Res 99 (1976)
Preliminary notes
Fig. 4. Examples of 33258 Hoechst fluorescence patterns illustrating terminal replication in mouse Y chromosomes. Cells were grown in medium containing BUdR except for a 3f h terminal pulse of deoxycytidine (A, B, C) or in control medium except for a 6-h terminal pulse of BUdR (D). Chromosome (A) exhibits dull fluorescence, presumably because a negligible amount of the deoxythymidine pulse had been incorporated. Chromosomes B and C exhibit slightly brighter fluorescence, more marked in the distal half of the chromosome. Chromosome D shows a complementary pattern, with dull fluorescence accentuated in the distal half of the chromosome. Note the lack of selective bright fluorescence in the centromerit regions consistent with the reported absence of mouse satellite DNA from the Y chromosome [33].
also permits additional differentiation within and between mouse chromosomes. In cells administered a 3f or a 6-h terminal pulse of TdR, bright fluorescence was usually localized to the centromeric regions and the late replicating X or Y chromosome (e.g. fig. 1b). Identification of deoxythymidine incorporation in centromeric areas was relatively unambiguous, since a single cycle of BUdR incorporation into these regions results in a lateral asymmetry of 33258 Hoechst fluorescence [23]. Bright fluorescence of an X or a Y was not observed in the absence of bright centromeres. However, in a few cells, a variable proportion of the centromeres exhibited bilaterally bright fluorescence, and chromosome arm fluorescence was uniformly dull. These results are consistent with previous reports [31, 321 that mouse satellite DNA, which is located in the centromeric regions [33], replicates late in S. They suggest that the
443
timing of satellite DNA replication may not be the same in all chromosomes. Hsu & Markvong [34] have similarly observed that very late replication in Mus muscufus, as determined by autoradiography, did not involve all centromere regions, even when grains were present over some of the chromosome arms. These authors obtained data with both autoradiography and a modified BUdR-Giemsa technique [ 171 indicating that late replication followed a banded distribution in the chromosome arms of Mus dunni. Similarly, we observed that in many cells administered a terminal deoxythymidine pulse (e.g. fig. 2c, 9-h pulse), extensive bright fluorescence occurred in a banded distribution in the autosome arms. This fluorescence pattern consists of a subset of the quinacrine bands of mouse chromosomes [29, 301, together with the C-band positive centromeres. Within the X, late replication is most pronounced in and around the centromeric region, and in a prominent band in the distal region of the chromosome arm (fig. 3). Unlike the results with human female lymphocytes [35, 361, qualitative differences in the internal order of replication between the two mouse X chromosomes have not thus far been observed. Late replication in the mouse Y usually appears to involve the entire chromosome. In an occasional cell, late replication is somewhat more pronounced in a localized region just distal to the middle of this chromosome (fig. 4). The technique of BUdR incorporation and staining with Hoechst 33258 for detecting late DNA replicating regions of chromosomes appears to be useful for detecting asynchronous replication of X and Y chromosomes in mice. Because of the high percentage of female cells showing a late replicating X and the sharp demarcation of the early and late replicating re-
444
Preliminary notes
gions, this technique should prove useful in examining X-autosome translocations [37] for possible position-dependent alterations in replication kinetics [38]. In addition, the method may be especially useful for detecting small fragments of late-replicating X or Y chromatin translocated to autosomal chromosomes [39]. This work was supported by a grant from the National Foundation March of Dimes (l-353) and a research career development award (GM 00122) from the National Institute of Genera1 Medical Sciences.
30. Miller, D A & Miller, 0 J. Science 178 f1972) 949. 31. Tobia, A, Schildkraut, C L & Maio, J J,‘J mdl biol 54 (1970) 499. 32. - Biochim biophys acta 246 (1971) 258. 33. Pardue, M L & Gall, J, Science 168(1970) 1356. 34. Hsu, T C & Markvong, A, Chromosoma 5 1 (1975)
_111.__.
35. Willard, H & Latt, S, Am j human genet (1976). In press. 36. Latt, S A, Exp cell res 86 (1974) 412. 37. y?, S & Cattanach, B M, Cytogenetics 1 (1%2) 38. Either, E, Adv genet 15 (1970) 175. 39. Cattanach, B M, Birth defects (ed A Motulsky & F Ebling) p. 129. Excerpta Medica, Amsterdam (1974). Received December 17, 1975 Accepted January 21, 1976
References Evans, H J, Ford, C E, Lyon, M F & Gray, J, Nature 206 (1965) 900. 2. Galton, M & Holt, S F, Exp cell res 37 (1965) 111. 3. Tiepolo, I, Fraccaro, M, Hulten, M, Lindsten, J, Mannini, A & Ming, P M L, Cytogenetics 6 (1%7) 51. 4. Nesbitt, M N & Gartler, S M, Cytogenetics 9 (1970) 212. 5. Craig, A P & Shaw, M W, Chromosoma 32 (1971) 364. 6. Caspersson, T, Lindsten, J, Lomakka, G, Msller, A & Zech, L, Int rev exp path01 11 (1973) 1. 7. Hsu, T C, Ann rev genet 7 (1974) 153. Kanda, N, Exp cell res 80 (1973) 463. ;: Takagi, N, Exp cell res 86 (1974) 127. 10. Latt, S A, Proc natl acad sci US 70 (1973) 3395. 11. - J histochem cytochem 22 (1974) 478. 12. Latt, S A, Juergens, L A, Stetten, G, Willard, H F & Scher, C D, J histochem cytochem 23 (1975) 493. 13. DutrilIaux, M B, Laurent, C, Couturier, J & LeJeune, J, Compt rend acad sci D 276 (1973) 3 175. 14. Kato, H, Nature 251 (1974) 70. 15. Takagi, N & Sasaki, M, Nature 256 (1975) 640. 16. Perry, P&Wolff, S, Nature 251 (1974) 156. 17. Korenberg, J & Freedlender, E, Chromosoma 48 (1974) 355. 18. Kim, M A, Humangenetik 25 (1974) 179. 19. Gratzner, H, Ingram, D, Leif, R D & Castro, A, Exp cell res 95 (1975) 88. 20. Latt, S A, Somatic cell genet 1 (1975) 293. 21. - Science 185 (1974) 74. 22. - Proc natl acad sci US 71 (1974) 3162. 23. Lin, M S, Latt, S A & Davidson, R L, Exp cell res 86 (1974) 392. 24. Latt, S A, Davidson, R L, Lin, M S & Gerald, P S, Exp cell res 87 (1974) 425. 2s. Angell, R & Jacobs, P A, Chromosoma 51 (1975) 301. 26. Meuth, M & Green, H, Cell 2 (1974) 109. 27. Hilwig, I & Gropp, A, Exp cell res 75 (1972) 122. 28. Loewe, H & Urbanietz, J, Arzneimittelforsch 24 (1974) 1927. 29. Francke, U & Nesbitt, M, Proc natl acad sci US 68 (1971) 2918. 1.
Exp Cell Res 99 (I 976)
Separation of Kupffer and endothelial cells of the rat liver by centrifugal elutriation D. L. KNOOK and E. CH. SLEYSTER, Znsriturefor Experimental lands
Gerontology
TNO, Rijswijk,
The Nether-
Isolated non-parenchymal cells from rat liver were separated by centrifugal elutriation into two fractions consisting of structurally intact Kupffer and endothelial cells with purities of 91 and 95 %, respectively. Purified Kupffer and endothelial cells showed nearly equal specific activities for the lysosomal enzyme acid phosphatase, whereas the specific activity of cathepsin D was about 3 times higher in Kupffer cells. It was calculated that a significant amount of the cathepsin D activity in the liver is present in the Kupffer cells.
Summary.
The two main cell types in the mammalian liver are parenchymal cells and sinusoidal lining cells. Although the latter cell group occupies only 3 % of the total liver volume in the young adult rat, at least one-third of all liver cells are sinusoidal cells [l]. The non-parenchymal cells in the sinusoidal area have usually been described as Kupffer cells. Electron microscopic observations, however, showed, in addition to Kupffer cells, the presence of a number of cell types such as endothelial cells, fat-storing cells, pit cells, monocytes and other white blood cells [2]. In view of their number and function, Kupffer and endothelial cells are by far the most important sinusoidal lining