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Action of glutaraldehyde and formaldehyde on segmentation mitoses
ACTIONOFGLUTARALDEHYDEANDFORMALDEHYDE ONSEGMENTATIONMITOSES Inhibition
of Spindle
und Astral
Fibres,
Cenrrospheres
Blocked
P. SENTEIN
SUMMARY Glutaraldehyde and formaldehyde act as antimitotic substances at lower concentrations and as fixatives at higher ones. The arrested mitosis is of the quinnline type (d-mitosis); it is characterized by the disappearance of all the spindle and astral fibres, by the immobilization of chromosomes in the equatorial region and by the blocking of centrospheres. These blocked centrospheres are devoid of axtral fibres. densified, and intensely stained; they do not diminish in volume at the end of mitosis. An explanation for this phenomenon is proposed: the binding of two or several microtubule subunits by one molecule of the active substance into the storage structures would prevent the discharge of these microtubule subunits and consequently the construction of microtubules. Colchicine-like substances bind only to one microtubule subunit and consequently storage structures cannot be formed. The relation between fixative and antimitotic properties is discussed. A gradient of sensibility to antimitotic action is observed: the mitose\ are more eaGly arrested in the animal than in the vegetative hemisphere.
During the segmentation of Pleurodele eggs, formaldehyde 0.04 M was found to be an antimitotic substance of a special type, which causes a ‘blocking’ of centrospheres, a total disappearance of all the spindle and astral fibres [IS] and an immobilization of metaphasic chromosomes in the equatorial region. These blocked centrospheres appear as spherical dense bodies on both sides of the chromosomes or near the telophase, telo-prophase and prophase nuclei; they are stained intensely by methylene blue, aniline blue, orange G or pyronin. This action of formaldehyde is similar to that of quinoline [9, 14, 15, 161; the latter is less active (0.46 M instead of 0.04 M), but also less toxic. The particular type of mitosis blocked by quinoline was first observed by Tjio & Le-
van [20, 211 and named d-mitosis (deviating type of mitosis). The metaphase with two blocked centrospheres is, as we have observed, only one particular case thereof. This blocking may be interpreted as being due to the storage of precursor material for the microtubules. In the normal second segmentation mitosis, the maximal volume of centrospheres (up to 17 pm in diameter) is reached at prometaphase, when the large asters (up to I mm in diameter) are at the end of their regression. At this time the spindle, which now begins to develop, is too small to utilize all this material immediately. The centrospheres have their minimal volume at telo-prophase, when the same material is completely utilized for the growth of asters. On the other hand, Exptl
Cd
Res 95 (1975)
0.05 Dissociation between mitosis and cytodieresis
M, 4 h
0.025
M. 4 h
0.0125
M. 4 h
0.0063
M. 4 h
(1.003
125 M. 3 h
Maximum 2 nuclei foor I blastomere
Maximum 7 nuclei f01 I blastomere
Maximum 3 nuclei for I blastomere
Maximum 6 nuclei f01 I blastomcre
hl;,,ln~un, X nuclei for I bla~tomere
Complete
Complete
Complete
Complete
Incomplete
Blocking of centrospheres
Constant
Constant
Constant
Frequent inconstant”
Evolution of chromosomes in karyomeres and nuclei
Only the
Only the
Near the poles or at equator
Near the poles or at equator
Near the poles of at equator
Disappearance spindle and astral fibres
of
Formation of chromosomes the nuclei Symptoms cytoxicity (I Prophase
near poles
near poles
but
Incon\tant
in
0
0
+
+
i
++
+
0
0
0
of
nuclei
without
centrospheres.
smaller
than
centrospheres blocked by quinoline at telo-prophase may have the same volume as at metaphase, as not all the material concentrated around the centrioles can be utilized by the asters, which are completely suppressed. When studied with the electron microblocked by scope, the centrospheres quinoline included an accumulation of dense bodies of finely fibrillar structure, which persisted later than in the normal mitotic cycle; this observation strengthens the interpretation given above (Sentein & Ates, unpublished). We infer that the dense bodies are constituted by precursors of microtubules, probably a special organization of microtubule subunits. Glutaraldehyde is also an agent of centrosphere blocking. As formaldehyde, it is a usual fixative and it is evident that the relation between the antimitotic and fixative properties of both substances- lies in their binding to proteins. But, when these substances are used as fixatives, it is always at
at higher
concentrations.
higher concentrations than when they act as antimitotics: in the first case they bind to all protein structures, in the second to the proteins of microtubules only. MATERIAL
AND METHODS
used eggs of Triturus hei~~ctiws Raz.. of Plrurodeles ~~.a/t/ii Michah.. Triturus tnarmorutus Laur., or Bu& h~fi, L.. which react in the same way. The formaldehyde was a 378 solution from E Merck A.G., Darmstadt and glutaraldehyde a 25% solution from Fluka A.G., Buchs S.G. The latter was purified and filtrated on Millipore. The eggs were treated before the first cleavage. at 2. 4, 8. 16 blastomeres and at various morula and blastula stages. The presence or absence of the jelly coat does not affect the results. All the experiments were performed at 18°C. The eggs were fixed, serially sectioned, and stained by our usual methods [7, 81. We have mostly and additionally
RESULTS The observations here reported were made on eggs of Triturus helveticus Raz. Glutaraldehyde
(1) Relations between the concentrations and the effects obtained. Five concentra-
Glutaraldehyde
and formaldehyde
as antimitotics
235
Table 2. Nuclei from eggs treated at two or four blastomeres, with 0.05 M concentration for 2 h, at different stages of the chromosome cycle, compared with normal nuclei
No. of nuclei at each phase in the treated eggs . .
Metaphases and anaphases blocked (chromosomes with 2 centrospheres)
Telophasic nuclei blocked (generally with I centrosphere for a nucleus or karyomere group each pole)
14
8
Metaphases anaphases No. of nuclei at each phase in the normal eggs . . .
II
at
Telo-prophasic nuclei blocked (generally with 2 centrospheres for a nucleus)
4
Total number nuclei
26
Telophases
Telo-prophases and prophases
Total number nuclei
3
15
29
and
tions were used: 0.05, 0.025, 0.0125, 0.00625, and 0.003125 M. The first one is cytotoxic after 2 h, its antimitotic action is the same after 2, 4, or 5 h. The second concentration (0.025 M) is less cytotoxic. The last ones do not destroy the astral fibres completely (fig. 30). There is little difference between the first two concentrations: 2 h of 0.05 M gives the same blocked mitoses as 4 h or 5 h of 0.025 M. The results are summarized in table 1. These results prompt some remarks: (a) The time-lag between the inhibition of cleavage and that of mitosis increases with the lowering of concentration, (b) The blocking of centrospheres is characterized by their densification and more intense staining, associated with the disappearance of the astral fibres (dense and smooth centrospheres). However, at the lowest concentration (0.003125 M) there may be neither fibres nor centrospheres. At 0.00625 M the blocked centrospheres are smaller than at higher concentrations; similar results suggest that in general the absence or reduction in volume of blocked
of
of
centrospheres is correlated to a weaker action; in that view, colchicine would be a weaker agent than quinoline with respect to its antimitotic action, if we compare their effects at the most active concentrations. (c) At the strongest concentration (0.05 M), in the scarce telophases observed, the transformation of chromosomes into karyomeres occurred near the poles, before the centrospheres are blocked, but not at the equator. At 0.0125 M and at lower concentrations, the chromosomes arrested in the equatorial plane may be transformed into karyomeres, as with quinoline [15] figs 30 and 64. At the intermediate concentration (0.025 M), only chromosomes which have initiated their .movement towards the poles may be transformed into karyomeres, with the result that the latter fill the space between the two blocked centrospheres (figs 14, 15). (d) The fusion of karyomeres into gonomeres does not seem to be inhibited by glutaraldehyde. However, the telo-prophasic nucleus is more or less prevented from becoming a prophasic one: at 0.05 and Exptl
Cell
Res 95 (1975)
236
P. Sentcilr
0.025 M there are no nuclei containing visible chromosomes in formation: the evolution is stopped at telophase and teloprophase (end of DNA synthesis), as can be seen in table 2. (e) The symptoms of cytotoxicity are: defibrillated achromatic material, nuclear sap intensely stained by methylene blue ot blue-black naphthol, some parts of the vitellus not normally stained, nuclei homogeneous, chromosomes altered and clumped. v) After 2 h of 0.05 M concentration the antimitotic action is strong, but incomplete: the spindle does not disappear completely: some of its fibres are always visible (see fig. 6).
(2) Number OJ‘ nuclei in LI hlastomere. Since the cleavage is arrested before mitosis, each blastomere has frequently at least two nuclei at telo-prophase and prophase (fig. 7). In our experimental conditions we have never observed two blocked metaphases in one blastomere-contrary to the observations made with the antimitotics of the colchicine type. From 40 eggs at two or four blastomeres, 28 have two nuclei to a blastomere and 12 only one. The number of nuclei required for a blastomere may increase at lower concentrations (table 1). (3) Relations oj’ centrospheres to the nuclei. The number of centrospheres required for a nucleus differs according to the phase of the mitotic cycle. At metaphase, anaphase, and early telophase, there are always two centrospheres to a blocked mitosis, therefore one for a polar group of chromosomes or karyomeres. At the end of telophase, telo-prophase, and true prophase, we may observe one or two centrospheres to a nucleus, or a group of karyomeres, according to the way they are blocked before or after division. Since in normal conditions this division occurs at the end of telophase, one must Exptl
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Res 95 (1975)
foresee that at this time there will be rather one than two centrospheres and rather two later (table 3). We may conclude from these observations that the centrospheres may be blocked at any moment before (figs 3. 7), during (fig. 8), or after their division (figs 9, 10, 11) and that they may appear divided (two blocked centrospheres) or subdivided (three or four centrospheres). due to the inception of the blocking action. They (one or two) may be either in contact with the nucleus (figs 4, 7, 9, 10, 1 I, 23, 24, 25, 30) or removed from it (fig. 16). In the latter case the distance between the centrospheres and the nucleus increases at telo-prophase and, moreover. at true prophase. In some cases the centrospheres may be at considerable distance from the nucleus: in one egg treated at two blastomeres by 0.025 M, for 5 h the distance was 48 pm, approximately half the length of the mitotic axis at anaphase. Sometimes one centrosphere is in contact with the nucleus and the other far from it. To explain this phenomenon we must discuss how the mitotic axis is normally lengthened during anaphase, telophase, and teloprophase. During anaphase the movement of chromosomes towards the poles is determined by the shortening of the
Figs 1-7. Glutarafdehyde 0.05 M 2 h. Figs 8-16. Glutaraldehyde 0.025 M 5 h. Fig. 17. Glutaraldehyde 0.025 M 4 h. Figs 18-2 1. Glutaraldehyde 0.025 M 2 h. Figs I, 2, 3, 4. Eggs treated at 4 blastomeres. Figs 5, 7, II, 20 and 21. Eggs treated at two blastomeres. Figs 6, 8, 9, 10. 12-15, 17. Morulas treated. Figs 16, 18, 19. Eggs treated at eight blastomeres. All the photographs were taken from sections stained with safranin, methylene blue, and orange G with a green filter. All are at the same scale (fig. I).
Glutaraldehyde
andformaldehyde
us antimitotics
Exptl
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Res 95 (1975)
One centrosphere to a nucleus I. Tclopllasic nuclei Eggs with 4 nuclei (2 or 4 blastomeres) Eggs with 8 nuclei (4 or 8 blastomeres) To!al no. of nuclei 2. Telo-prophusic und praphasic nuclei Eggs with 2 nuclei (1 or 2 blastomeres) Eggs with 4 nuclei (2 or 4 blastomeres) Eggs with 8 nuclei (4 or 8 blastomeres) Total
no. of nuclei
Two centrospheres to a nucleus
Cell Res 95 (1975)
Total no. of nuclei
44
6
0
50
26 70
0 6
0 0
26 76
0
2
0
2
6
40
0
46
6
?I
3
30
I2
66
chromosomal (kinetochore) fibres, which may be helped by the lengthening of the pole-to-pole fibres. But after the end of anaphase the spindle cannot be responsible for the lengthening of the distance between poles, since it regresses in its equatorial part and separates into two halves. At this time, only a pulling by the astral fibres, which are anchored to the cortex and shortened by their proximal ends, can explain the increasing distance between the centrospheres, which in that way are retracted towards the cortex. A similar mechanism may explain the separation of the newly formed poles for the subsequent mitosis. Since the shortening of the astral fibres is a depolymerization of microtubules, it cannot be inhibited by an antimitotic substance. On the contrary, since the lengthening of the spindle at the beginning of anaphase is a polymerization phenomenon, it may be arrested and the chromosomes may stop at some distance from the poles. If these chromsomes become karyomeres and Exptl
More than two centrospheres to a nucleus
78
then nuclei in such an abnormal position and if, in the meantime, the poles are retracted by the shortening of the astral fibres, the separation of centrospheres and
Fig. 22. Morula treated by glutaraldehyde 0.025 M for 2 h. Staining: methyl green, pyronin. Figs 23-27. Two blastomeres. Eggs treated with glutaraldehyde 0.0125 M for 6 h. Staining: safranin, methyl blue, orange G. Green filter. Figs 28,29. Two sections of the same amphimixy with two blocked centrospheres. Glutaraldehyde 0.05 M for 2 h. Methyl green, pyronin. Green filter. Fig. 30. Eight blastomere egg treated with glutaraldehyde 0.003125 M for 4 h. Safranin, methylene blue, orange G. Red filter. Fig. 31. Uncleaved egg treated with glutaraldehyde 0.00625 M for 4 h. Same staining. Red filter. Figs 22-31. Eggs of Triturus helveticus Raz. Fig. 32. Morula of Pleurodeles waltlii Michah., treated with glutaraldehyde 0.025 M for 6 h. Same staining. Green filter. Figs 33-36. Eight blastomeres. Egg of Triturus treated with formaldehyde 1 M for 2 h. Figs 37, 38. Eight blastomeres. Egg of Triturus. Formaldehyde 0.1 M for 2 h. Fig. 39. Four blastomeres. Egg of Triturus treated with formaldehyde 0.05 M for 2 h. Fig. 40. Morula of Pleurodeles. Formaldehyde 0.1 M for 10 h. Excepted figs 37, 38 and 39, all the figures at the same magnification as fig. 22.
Gluturaldehyde
andformaldehyde
as antimitotics
Exptl
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239
Res 95 (1975)
240
P. Sentein
nuclei may occur. This explanation presupposes that the lengthening of the spindle is inhibited before destruction of the astral fibres commences, a development which occurs only when these astral fibres begin to expand. (4) Morphology oj’ the blocked mitoses and nuclei. Let us now consider some mor-
phological aspects of the mitoses and nuclei after 0.05 M for 2 h (figs l-7). The types of centrospheres differ according to the stage of mitosis: at metaphase they are granular, not homogeneous (figs I, 2), and ringshaped (fig. 2); between them and the chromosomes appears an achromatic material, incompletely defibrillated (figs 1, 2, 6). In some cases, spindle fibres are still visible and distorted (fig. 6). Chromosomes are in the equatorial region, perpendicular or slightly oblique to the mitotic axis. In some cases the centrospheres appear crescent-shaped in longitudinal section. the achromatic material disappears and the chromosomes are clumped (fig. 5). At telophase, karyomeres are near the centrosphere, but not oriented towards the pole and the single centrosphere is dense, spherical and devoid of astral fibres (fig. 3). This difference between granular and dissociated centrospheres at metaphase, and dense homogeneous centrospheres at telophase, occurs only at strong concentrations and with brief treatment. At telo-prophase the single, densified centrospheres may have a more irregular form (fig. 4). Each of the two nuclei (in fig. 7) in the same blastomere has two centrospheres, but only one is visible on the section. Paradoxically, after 2 h, at a concentration of 0.05 M, the spindle material seems to have been better preserved than at 0.025 M: in the latter case it is completely absent, the centrospheres are nearer each other and Exptl
Cell
Res 95 (1973
more condensed (fig. 1X. to be compared with figs 1, 2 and ’ 6). This better preservation of the achromatic apparatus at strong concentration is an effect of partial fixation. After 0.025 M for 4 or 5 h (figs 8 to 17), at all the phases of mitosis, the fibres disappear completely and the centrospheres are constantly blocked. The latter may be in the process of subdivision (fig. 8). Two centrospheres may be in contact with the nucleus, but not exactly opposite each other (figs 9 and 10, two adjacent sections). In fig. I I, from a morula treated in the same manner, two centrospheres (arrow heads), approximately of the same volume. are at the opposite poles of the nucleus. Chromosomes may be assembled in the equatorial region, but not exactly in the equatorial plane; in figs 12 and 13, two sections from the same blocked mitosis, the centrospheres are on both sides of the chromosome group; that may be considered as a beginning of anaphase. At telophase the karyomeres have the same disposition as the chromosomes at meta-anaphase with regard to the centrospheres: they are not exactly in the equatorial plane (figs 14 and 15, two adjacent sections of the same mitosis). In the prophase of fig. 16 the small nucleus contains fine threads and chromosomes; the two blocked centrospheres are remote from it. Fig. 17 shows a metaphase from a two blastomeres egg treated with 0.025 M concentration for 4 h: the chromosomes are thickened and clumped, but with a shorter treatment of the same solution (2 h, egg treated at eight blastomeres) they are thinner and may be easily distinguished, although not of a normal shape (fig. 18). The same applies for an egg at two blastomeres after 2 h of treatment (fig. 19). The three metaphases of figs 17, 18 and 19 are good examples of the quinoline-like blocked
Glutaraldehyde
mitoses, each with two spherical, densified centrospheres, strongly stained by methyl blue on both sides of the chromosomes. They are also intensely stained by pyronin with the method of Unna (fig. 22), but not if previously treated with ribonuclease. This presence of RNA must be related to the great number of ribosomes observed with the electron microscope in the centrospheres blocked by quinoline (Sentein & Ates (1974), unpublished). Probably the same applies for glutaraldehyde. With the same treatment of a two blastomeres egg for 2 h, the two centrospheres at telophase are of the same type (figs 20 and 21, two adjacent sections of the same telophase): they are spherical, equal to or longer than the centrospheres at metaphase of figs 17, 18, 19. Note that the karyomeres of the left pole (fig. 20) are beyond it; in this case they have overshot the mark. There is only one centrosphere for a group of karyomeres, because the blocking has occurred before its division. From these observations on the action of 0.05 M and 0.025 M concentrations we may note that centrospheres blocked at metaphase may have two types of structure, granular or condensed, while at telophase, telo-prophase, and prophase, they are always condensed and homogeneous. In the latter case they may be formed by a peripheral, intensely stained zone, annular or crescent-shaped, and a less intensely stained central zone. These ‘empty’ centrospheres are more often observed at the lower concentrations (0.0125 M and 0.00625 M). At 0.003125 M, centrospheres may or may not be blocked. The same observations were made with the use of quinoline [14, 15, 161. After 0.0125 M for 6 h, the centrospheres are always blocked and often the nuclei are upper in the animal hemisphere. They begin
andJbrmaldehyde
US rrntimitotics
241
towards the cortex a motion which is less accentuated than with the antimitotics of the colchicine type. In figs 23, 24 and 25 the single centrosphere is near the end of the nucleus, and not in the middle of one of its sides (fig. 23). In figs 26 and 27 (two adjacent sections) the telo-prophasic polyploid nucleus results from the fusion of two groups of karyomeres (as in figs 14 and 15). Consequently the centrospheres are on the same side (arrow head) and not at the opposite poles of the nucleus, as in fig. 1I. The concentration 0.003125 M is the threshold where the fibrillae reappear and the blocking is not constant. A telo-prophasic nucleus from this experiment is shown in fig. 30: some short fibres are preserved around the blocked centrosphere. The action of all the concentrations used was particularly interesting at the time of amphimixy. The centrospheres were always completely blocked, even at the lowest concentrations; astral fibrillae were never formed: this stage is the most sensitive to defibrillation, perhaps because it corresponds to a lengthened telo-prophase. Here also the blocking may occur before or after the division of the centrosphere: in figs 28 and 29 (two adjacent sections of the same amphimixy after 0.05 M for 2 h, stained with methyl green-pyronin) there are two blocked centrospheres at the junction of the pronuclei; opposite to the normal amphimixy, they are not joined by centrodesmotic fibres. But in other cases and especially in fig. 31 (0.00625 M for 4 h) only one centrosphere exists. Similar observations were made with quinoline. The important conclusion of this fact is that under normal conditions there is one division of the centrosphere at the time of amphimixy, that is to say, a separation of the centrioles which are included into it and not a reunion of two centrioles, which might Exprl
Cd
Res 95 (1975)
242
P. Sctitcin
be brought, the one by the spermatozoon and the other by the oocyte. Action of‘gluturuldehyde on the diastemtr The segmentation mitoses are characterized by a special structure, similar to the phragmoplast of the plant cell, which was named diastema; it is fibrillar and situated half-way between the two telophasic nuclei. With glutaraldehyde, some two blastomere eggs may contain four teloprophasic nuclei; between two of them a fibrillar diastema may persist, which indicates the plane which would be normally occupied by the furrow. However, it does not become strongly hyperfibrillar, as under the action of carboxylic acids [13, 191. (5) The action on eggs oj’ Pleurodeles and Bufo. The action of glutaraldehyde on the eggs of Pleurodeles, other species of Triturus and Bufb, does not differ from that on the egg of Triturus helveticus Raz. For instance, from a morula of Pleurodeles waltlii Michah., fig. 32 shows a telo-prophasic nucleus asymmetrically pinched between the two blocked centrospheres. Formaldehyde
Formaldehyde is used as a fixative at 20% of a 37 % commercial solution (2.46 M). The molar concentration has the same effect and, as we shall see, this effect remains partial with 0. I M. The antimitotic effect is observed at 0.1, 0.05,0.02, and 0.025 M (2 h) but 0.0125 M is inactive. The active concentrations being to be toxic after 2 h, sooner than with glutaraldehyde, and produce cytolysis. The formaldehyde has a narrower scale of utilization than glutaraldehyde and the fixation effect overlays the antimitotic one. 2.46 M and M concentrations of formaldehyde give mitotic figures no different in Expti
Cell Res 95 (1975)
size than the normal ones; the achromatic material, clearly delineated from hyaloplasm, is normal in quantity. but the astral and spindle fibres are not preserved (figs 33 to 36). If we compare eggs fixed with 2.46 M formaldehyde solution and post-fixed with Moricard fluid [7], with eggs of the same type fixed from the beginning by Moricard only, we observe that the latter have well preserved spindle and astral fibres, whereas the former have no fibres at all and that the equatorial part of the spindle is not fixed, while in the eggs only fixed by Moricard. this part is formed by conspicuous, well stained fibres. If the sections of eggs fixed with formaldehyde do not show well preserved spindle fibres, it is not because they were not stabilized and subsequently altered by technical manipulations, for instance by alcohols or toluene, but because they are actively destroyed by formaldehyde. Another paradox is that at strong fixative concentration (2.46 M) the centrospheres are granular at metaphase (figs 33-35), while they are homogeneous and more condensed in the eggs of the same stage with 0.1 M after 2 h (figs 37-38); in the latter case the action is mostly antimitotic. For glutaraldehyde, the centrospheres were granular at 0.05 M after 2 h (figs 1, 2, 6) and homogeneous at 0.025 M after the same time, but in the latter case spindle fibres were more destroyed than with the corresponding concentration of formaldehyde (0.1 M for 2 h) and the centrospheres were nearer each other. To explain such an observation, one must think that with formaldehyde at antimitotic concentrations the fixation effect persists longer than with glutaraldehyde. In the particular case of fig. 39, beyond the two blocked centrospheres (arrowheads), secondary poles seem to be formed
Glutaraldehyde
andformaldehyde
as antimitotics
243
PIommL c!ia MIC--suBuL~ITs
depolymerization polymerization
not prevented, prevented at all
levels
l
l.
0 l l oQJlNoLIl+LIKE
-893
progressive microtubules, thereafter
-
birding
to subunits
suEsTz4~cE
m
disappearance of dense bodies and centrospheres .
depolymerization not prevented, poiymerieation prevented at the level of microtubules
c@ 8 00 bin3.ngofsubunit.s or of mre cxxnplex structures between them Probably structures Fig. 41. Possible mechanism types of spindle inhibitors.
a direct wliich
transformtion may be added
of the action
to
The linking of molecules preserves or perhaps strengthens the storage or packaging structures (dense k&ies) of link&l s&units those previously
into formed.
storage
or
packagi-
for the two
Exptl
Cell Res 95 (1975)
(arrows), where normal poles would take place. We explain this fact by a tendency to form again new poles at the limit of the ‘peripheral network’ of the normal metaphase, as we have seen with selenium dioxide [ 1 I] (figs 8, 9). The action of formaldehyde 0. I M on eggs of Pleum<’.s \jxtltlii Michah., is absolutely the same as that on Triturrrs eggs (fig. 40).
At the lower active concentrations (0.02 and 0.025 M) mitoses are more completely blocked in the animal than in the vegetawhere normal bipolar tive blastomeres, mitoses may be observed. There is a grddient brought to light by the antimitotic activity of formaldehyde. A similar gradient was observed in the action of organomercurials (Sentein, unpublished). At 0.025 M after 2 h, centrospheres are completely blocked in the first mitosis of undivided eggs, which were treated before the first cleavage or before the second; on the contrary, the polarity is absolutely normal when treatment begins at four blastomeres or later. Mitosis seems to be protected where and when differentiation begins. DISCUSSION The difference between d-mitosis (quinoline) and c-mitosis (colchicine, vincaleukoblastine, podophyllin) was primarily explained as an effect of fixation by quinoline [20, 211. But even if such an effect exists at higher concentrations, it cannot explain the immobilization of chromosomes at the equator at lower concentrations, since these chromosomes may be transformed into karyomeres without changing place. The only possible explanation is the absence of fibres (microtubules). We have found three additional differences: with colchicine and colcemid the segmentation Exprl
Cell
Res 95 (1975)
mitoses are not immediately ,~rrestecl. hut always after at least one mitotic cycle. which is absolutely normal. Secondly. the centrospheres disappear, at least at the light microscope level [4]. Thirdly. blocked mitoses and nuclei go up towards the cortex and/or the animal pole. The same is true of vincaleukoblastin [IO]. On the other hand, the action of quinoline, glutaraldehyde and formaldehyde is immediate and may appear at the first cycle after treatment; mitosis and cleavage are simultaneously arrested and two metaphases are never seen in one blastomere; secondly, these substances produce the ‘blocking’ of centrospheres. There are no conspicuous centrospheres outside the segmentation mitoses. In the normal second segmentation mitosis they reach I7 pm in diameter at prometaphase. In 8 blastomere eggs. blocked centrospheres at metaphase are smaller than the normal ones, 8 instead of I I pm. but the diameter of blocked centrospheres at telophase and telo-prophase may be greater than in normal ones, since they do not dissociate, as in normal conditions: consequently they are roughly similar at metaphase and at telo-prophase. Similar observations are easily explained if we consider that astral regression is the principal source of subunits which furnish the storage structures of the centrospheres. Normally the so-packaged material is utilized at the end of mitosis for astral development. But here, the latter is inhibited and the precursor material remains around the centriole. other material (riboSimultaneously somes and glycogen granules. vesicles, mitochondria and other organelles) may be retained in the centrospheres and remain accumulated therein. Their volume depends on that accumulation. The presence of ARN (pyronin staining suppressed by
Glutaraidehyde RNase) and glycogen (PAS positive) into centrospheres, blocked or not, is evident. The blocked centrospheres without any spindle or astral fibres may be observed not only with quinoline, glutaraldehyde and formaldehyde, but also: (u) with some alkylating agents: nitrogen mustard l/l 000 after 6 h [2, 31 and ethyleneimine-quinones [6]; in the first case no chromosome breakage appears when mitosis is blocked; (6) with organo-mercurials and N-ethylmaleimide, two substances which bind to -SH groups of the proteins; (c) with phenol M/l 000 ([5] figs 1, 2) and amphetamine sulfate M/800 ([7,8] figures l-10,37). The centrospheres may be blocked in the presence of spindle fibres, but always without astral fibres by some carboxylic acids [12, 131. In some cases the tips of the spindle are narrowed and often bent back or coiled ([19] figures 3, 12,35, 39,41, 54). In summary, blocked centrospheres may be observed with a more or less abnormal spindle, but never when spindle and asters persist simultaneously. We must therefore attribute the greatest importance to the construction of asters in order to explain the normal telophasic reduction of centrospheres, and the inhibition of asters to explain their blocking. The nature of this phenomenon will only be understood when an extensive study of the ultrastructure of blocked centrospheres is completed. This study is most advanced for quinoline (Sentein & Ates, unpublished) and it is evident that the blocked telophasic centrospheres include many dense and striated bodies, accumulated at a phase when they are normally absent, and many ribosomes and glycogen granules. The first few results we have obtained with glutaraldehyde (Sentein & Ates, unpublished), are of the same type. The relations between fixation effect and
and formaldehyde
as antimitotics
245
antimitotic action can be explained: in the first case all the cellular structural proteins are linked to many fixative molecules; in the second case only the heterodimeres of tubulin are linked to antimitotic molecules: it is not surprising that more molecules are needed in the first case. But it is evident from our observations that the fixation effect may cloud the consequences of the antimitotic action (though not completely), with formaldehyde. Glutaraldehyde is a better fixative than formaldehyde and exerts its antimitotic action over a larger scale of concentrations. Moreover the pure antimitotic action is observed for the latter at the lower active concentration (0.025 M) and the total disappearance of spindle fibres only in some cases (e.g. fig. 39). On the other hand, even with glutaraldehyde, not all the fibres of the achromatic apparatus in the cleaving egg are as well conserved as with the Moricard fixative. Our observations may be brought nearer to the 50 % reduction of spindle birefringence in the oocytes of Pectinaria by glutaraldehyde, as by colcemid [I]. Acetic acid, which is often associated with formaldehyde in fixative mixtures, namely in Moricard fluid, is known to preserve the spindle fibres very well, because its antimitotic action is different from that of formaldehyde: we have observed that it dissociates the spindle and astral fibres, but usually does not destroy them (Sentein, unpublished). The association of the two substances in suitable concentrations may correct the disadvantage of each of them and give a good compromise for fixation, as observed by all cytologists. Although glutaraldehyde is a better fixative than formaldehyde, its action on the cytoplasm is of the same kind and it is doubtful whether it preserves all the microExprlCcl/
Rrs 95 (1975)
246
P.
Soltc~itr
tubular structures. Comparison at the light microscope level of half thick sections fixed by glutaraldehyde, with sections fixed by Moricard fluid, is meaningful in this respect. One possible explanation of the differences between the action of colchicine-like and quinoline-like substances is given in fig. 41. In the normal segmentation mitosis, microtubule subunits are packaged into complex, finely fibrillar structures, which constitute dense bodies and probably striated bodies. One molecule of colchicine binds only to one heterodimere of tubulin: the polymerization of microtubules and the formation of storage structures are then simultaneously prevented. On the other hand, to explain the persistence and accumulation of these structures in the centrospheres blocked by quinoline we may suppose a binding of two or several microtubule subunits between them by one molecule of quinoline; these structures would then be stabilized and consequently the polymerization of microtubules would be prevented, as also the breaking up of storage structures. In our first electron microscope observations, glutaraldehyde seems to have had the same effects as quinoline, accumulating dense bodies in the blocked centrospheres (Sentein & Ates, unpub-
Exptl
Cell
Res 95 (1975)
lished). The same explanation may be proposed to apply to both substances. The fixation must be considered an experimental procedure. as the antimitotic action, and the molecular aspects of these two actions, must be studied in the same way. We acknowledge the technical assistance of Huguette Niel. This work was supported by grants from the CNRS, the Fondation pour la recherche medicale francaise, and by a contract from INSERM (no. 72-l-012-1).
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. II. 12. 13. 14. 15. 16. 17. IS. 19.
Inoue, S & Sato. H, J gen physiol 50 (1967) 259. Sentein, P. Compt rend sot biol 149 (1955) 167. Compt rend assoc anat 42 (1955) 1238. Arch anat microsc 45 (1956) 99. Acta anat 34 (1958) 201. Colloq int centre natl rech sci 88 (1960) 143. Chromosoma I3 (1962) 67. Acta anat 49 (1962) 297. Compt rend acad sci 256 (1963) 4759 Chromosoma IS (1964) 416. Ibid 17 (1965) 336. Compt rend acad sci 267 (1968) 1895. Chromosoma 24 (1968) 67. Bull assoc anat 55 (1970) 973. Chromosoma 32 (1970) 97. Compt rend sot biol 164 (1970) 1562. Bull assoc anat 55 (1970) 973. Ibid 56 (1971) 712. Sentein, P & Vannereau, H, Chromosoma 40 (1973) I. 20. Tjio, J H & Levan, A, Anal estac exp aula dei 2 (1950) 21. 21. Lunds Univ arsskr p. I. (1954). Received Revised
January version
3 I, 1975 received April
I, 1975
of Spindle
und Astral
Fibres,
Cenrrospheres
Blocked
P. SENTEIN
SUMMARY Glutaraldehyde and formaldehyde act as antimitotic substances at lower concentrations and as fixatives at higher ones. The arrested mitosis is of the quinnline type (d-mitosis); it is characterized by the disappearance of all the spindle and astral fibres, by the immobilization of chromosomes in the equatorial region and by the blocking of centrospheres. These blocked centrospheres are devoid of axtral fibres. densified, and intensely stained; they do not diminish in volume at the end of mitosis. An explanation for this phenomenon is proposed: the binding of two or several microtubule subunits by one molecule of the active substance into the storage structures would prevent the discharge of these microtubule subunits and consequently the construction of microtubules. Colchicine-like substances bind only to one microtubule subunit and consequently storage structures cannot be formed. The relation between fixative and antimitotic properties is discussed. A gradient of sensibility to antimitotic action is observed: the mitose\ are more eaGly arrested in the animal than in the vegetative hemisphere.
During the segmentation of Pleurodele eggs, formaldehyde 0.04 M was found to be an antimitotic substance of a special type, which causes a ‘blocking’ of centrospheres, a total disappearance of all the spindle and astral fibres [IS] and an immobilization of metaphasic chromosomes in the equatorial region. These blocked centrospheres appear as spherical dense bodies on both sides of the chromosomes or near the telophase, telo-prophase and prophase nuclei; they are stained intensely by methylene blue, aniline blue, orange G or pyronin. This action of formaldehyde is similar to that of quinoline [9, 14, 15, 161; the latter is less active (0.46 M instead of 0.04 M), but also less toxic. The particular type of mitosis blocked by quinoline was first observed by Tjio & Le-
van [20, 211 and named d-mitosis (deviating type of mitosis). The metaphase with two blocked centrospheres is, as we have observed, only one particular case thereof. This blocking may be interpreted as being due to the storage of precursor material for the microtubules. In the normal second segmentation mitosis, the maximal volume of centrospheres (up to 17 pm in diameter) is reached at prometaphase, when the large asters (up to I mm in diameter) are at the end of their regression. At this time the spindle, which now begins to develop, is too small to utilize all this material immediately. The centrospheres have their minimal volume at telo-prophase, when the same material is completely utilized for the growth of asters. On the other hand, Exptl
Cd
Res 95 (1975)
0.05 Dissociation between mitosis and cytodieresis
M, 4 h
0.025
M. 4 h
0.0125
M. 4 h
0.0063
M. 4 h
(1.003
125 M. 3 h
Maximum 2 nuclei foor I blastomere
Maximum 7 nuclei f01 I blastomere
Maximum 3 nuclei for I blastomere
Maximum 6 nuclei f01 I blastomcre
hl;,,ln~un, X nuclei for I bla~tomere
Complete
Complete
Complete
Complete
Incomplete
Blocking of centrospheres
Constant
Constant
Constant
Frequent inconstant”
Evolution of chromosomes in karyomeres and nuclei
Only the
Only the
Near the poles or at equator
Near the poles or at equator
Near the poles of at equator
Disappearance spindle and astral fibres
of
Formation of chromosomes the nuclei Symptoms cytoxicity (I Prophase
near poles
near poles
but
Incon\tant
in
0
0
+
+
i
++
+
0
0
0
of
nuclei
without
centrospheres.
smaller
than
centrospheres blocked by quinoline at telo-prophase may have the same volume as at metaphase, as not all the material concentrated around the centrioles can be utilized by the asters, which are completely suppressed. When studied with the electron microblocked by scope, the centrospheres quinoline included an accumulation of dense bodies of finely fibrillar structure, which persisted later than in the normal mitotic cycle; this observation strengthens the interpretation given above (Sentein & Ates, unpublished). We infer that the dense bodies are constituted by precursors of microtubules, probably a special organization of microtubule subunits. Glutaraldehyde is also an agent of centrosphere blocking. As formaldehyde, it is a usual fixative and it is evident that the relation between the antimitotic and fixative properties of both substances- lies in their binding to proteins. But, when these substances are used as fixatives, it is always at
at higher
concentrations.
higher concentrations than when they act as antimitotics: in the first case they bind to all protein structures, in the second to the proteins of microtubules only. MATERIAL
AND METHODS
used eggs of Triturus hei~~ctiws Raz.. of Plrurodeles ~~.a/t/ii Michah.. Triturus tnarmorutus Laur., or Bu& h~fi, L.. which react in the same way. The formaldehyde was a 378 solution from E Merck A.G., Darmstadt and glutaraldehyde a 25% solution from Fluka A.G., Buchs S.G. The latter was purified and filtrated on Millipore. The eggs were treated before the first cleavage. at 2. 4, 8. 16 blastomeres and at various morula and blastula stages. The presence or absence of the jelly coat does not affect the results. All the experiments were performed at 18°C. The eggs were fixed, serially sectioned, and stained by our usual methods [7, 81. We have mostly and additionally
RESULTS The observations here reported were made on eggs of Triturus helveticus Raz. Glutaraldehyde
(1) Relations between the concentrations and the effects obtained. Five concentra-
Glutaraldehyde
and formaldehyde
as antimitotics
235
Table 2. Nuclei from eggs treated at two or four blastomeres, with 0.05 M concentration for 2 h, at different stages of the chromosome cycle, compared with normal nuclei
No. of nuclei at each phase in the treated eggs . .
Metaphases and anaphases blocked (chromosomes with 2 centrospheres)
Telophasic nuclei blocked (generally with I centrosphere for a nucleus or karyomere group each pole)
14
8
Metaphases anaphases No. of nuclei at each phase in the normal eggs . . .
II
at
Telo-prophasic nuclei blocked (generally with 2 centrospheres for a nucleus)
4
Total number nuclei
26
Telophases
Telo-prophases and prophases
Total number nuclei
3
15
29
and
tions were used: 0.05, 0.025, 0.0125, 0.00625, and 0.003125 M. The first one is cytotoxic after 2 h, its antimitotic action is the same after 2, 4, or 5 h. The second concentration (0.025 M) is less cytotoxic. The last ones do not destroy the astral fibres completely (fig. 30). There is little difference between the first two concentrations: 2 h of 0.05 M gives the same blocked mitoses as 4 h or 5 h of 0.025 M. The results are summarized in table 1. These results prompt some remarks: (a) The time-lag between the inhibition of cleavage and that of mitosis increases with the lowering of concentration, (b) The blocking of centrospheres is characterized by their densification and more intense staining, associated with the disappearance of the astral fibres (dense and smooth centrospheres). However, at the lowest concentration (0.003125 M) there may be neither fibres nor centrospheres. At 0.00625 M the blocked centrospheres are smaller than at higher concentrations; similar results suggest that in general the absence or reduction in volume of blocked
of
of
centrospheres is correlated to a weaker action; in that view, colchicine would be a weaker agent than quinoline with respect to its antimitotic action, if we compare their effects at the most active concentrations. (c) At the strongest concentration (0.05 M), in the scarce telophases observed, the transformation of chromosomes into karyomeres occurred near the poles, before the centrospheres are blocked, but not at the equator. At 0.0125 M and at lower concentrations, the chromosomes arrested in the equatorial plane may be transformed into karyomeres, as with quinoline [15] figs 30 and 64. At the intermediate concentration (0.025 M), only chromosomes which have initiated their .movement towards the poles may be transformed into karyomeres, with the result that the latter fill the space between the two blocked centrospheres (figs 14, 15). (d) The fusion of karyomeres into gonomeres does not seem to be inhibited by glutaraldehyde. However, the telo-prophasic nucleus is more or less prevented from becoming a prophasic one: at 0.05 and Exptl
Cell
Res 95 (1975)
236
P. Sentcilr
0.025 M there are no nuclei containing visible chromosomes in formation: the evolution is stopped at telophase and teloprophase (end of DNA synthesis), as can be seen in table 2. (e) The symptoms of cytotoxicity are: defibrillated achromatic material, nuclear sap intensely stained by methylene blue ot blue-black naphthol, some parts of the vitellus not normally stained, nuclei homogeneous, chromosomes altered and clumped. v) After 2 h of 0.05 M concentration the antimitotic action is strong, but incomplete: the spindle does not disappear completely: some of its fibres are always visible (see fig. 6).
(2) Number OJ‘ nuclei in LI hlastomere. Since the cleavage is arrested before mitosis, each blastomere has frequently at least two nuclei at telo-prophase and prophase (fig. 7). In our experimental conditions we have never observed two blocked metaphases in one blastomere-contrary to the observations made with the antimitotics of the colchicine type. From 40 eggs at two or four blastomeres, 28 have two nuclei to a blastomere and 12 only one. The number of nuclei required for a blastomere may increase at lower concentrations (table 1). (3) Relations oj’ centrospheres to the nuclei. The number of centrospheres required for a nucleus differs according to the phase of the mitotic cycle. At metaphase, anaphase, and early telophase, there are always two centrospheres to a blocked mitosis, therefore one for a polar group of chromosomes or karyomeres. At the end of telophase, telo-prophase, and true prophase, we may observe one or two centrospheres to a nucleus, or a group of karyomeres, according to the way they are blocked before or after division. Since in normal conditions this division occurs at the end of telophase, one must Exptl
Cell
Res 95 (1975)
foresee that at this time there will be rather one than two centrospheres and rather two later (table 3). We may conclude from these observations that the centrospheres may be blocked at any moment before (figs 3. 7), during (fig. 8), or after their division (figs 9, 10, 11) and that they may appear divided (two blocked centrospheres) or subdivided (three or four centrospheres). due to the inception of the blocking action. They (one or two) may be either in contact with the nucleus (figs 4, 7, 9, 10, 1 I, 23, 24, 25, 30) or removed from it (fig. 16). In the latter case the distance between the centrospheres and the nucleus increases at telo-prophase and, moreover. at true prophase. In some cases the centrospheres may be at considerable distance from the nucleus: in one egg treated at two blastomeres by 0.025 M, for 5 h the distance was 48 pm, approximately half the length of the mitotic axis at anaphase. Sometimes one centrosphere is in contact with the nucleus and the other far from it. To explain this phenomenon we must discuss how the mitotic axis is normally lengthened during anaphase, telophase, and teloprophase. During anaphase the movement of chromosomes towards the poles is determined by the shortening of the
Figs 1-7. Glutarafdehyde 0.05 M 2 h. Figs 8-16. Glutaraldehyde 0.025 M 5 h. Fig. 17. Glutaraldehyde 0.025 M 4 h. Figs 18-2 1. Glutaraldehyde 0.025 M 2 h. Figs I, 2, 3, 4. Eggs treated at 4 blastomeres. Figs 5, 7, II, 20 and 21. Eggs treated at two blastomeres. Figs 6, 8, 9, 10. 12-15, 17. Morulas treated. Figs 16, 18, 19. Eggs treated at eight blastomeres. All the photographs were taken from sections stained with safranin, methylene blue, and orange G with a green filter. All are at the same scale (fig. I).
Glutaraldehyde
andformaldehyde
us antimitotics
Exptl
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Res 95 (1975)
One centrosphere to a nucleus I. Tclopllasic nuclei Eggs with 4 nuclei (2 or 4 blastomeres) Eggs with 8 nuclei (4 or 8 blastomeres) To!al no. of nuclei 2. Telo-prophusic und praphasic nuclei Eggs with 2 nuclei (1 or 2 blastomeres) Eggs with 4 nuclei (2 or 4 blastomeres) Eggs with 8 nuclei (4 or 8 blastomeres) Total
no. of nuclei
Two centrospheres to a nucleus
Cell Res 95 (1975)
Total no. of nuclei
44
6
0
50
26 70
0 6
0 0
26 76
0
2
0
2
6
40
0
46
6
?I
3
30
I2
66
chromosomal (kinetochore) fibres, which may be helped by the lengthening of the pole-to-pole fibres. But after the end of anaphase the spindle cannot be responsible for the lengthening of the distance between poles, since it regresses in its equatorial part and separates into two halves. At this time, only a pulling by the astral fibres, which are anchored to the cortex and shortened by their proximal ends, can explain the increasing distance between the centrospheres, which in that way are retracted towards the cortex. A similar mechanism may explain the separation of the newly formed poles for the subsequent mitosis. Since the shortening of the astral fibres is a depolymerization of microtubules, it cannot be inhibited by an antimitotic substance. On the contrary, since the lengthening of the spindle at the beginning of anaphase is a polymerization phenomenon, it may be arrested and the chromosomes may stop at some distance from the poles. If these chromsomes become karyomeres and Exptl
More than two centrospheres to a nucleus
78
then nuclei in such an abnormal position and if, in the meantime, the poles are retracted by the shortening of the astral fibres, the separation of centrospheres and
Fig. 22. Morula treated by glutaraldehyde 0.025 M for 2 h. Staining: methyl green, pyronin. Figs 23-27. Two blastomeres. Eggs treated with glutaraldehyde 0.0125 M for 6 h. Staining: safranin, methyl blue, orange G. Green filter. Figs 28,29. Two sections of the same amphimixy with two blocked centrospheres. Glutaraldehyde 0.05 M for 2 h. Methyl green, pyronin. Green filter. Fig. 30. Eight blastomere egg treated with glutaraldehyde 0.003125 M for 4 h. Safranin, methylene blue, orange G. Red filter. Fig. 31. Uncleaved egg treated with glutaraldehyde 0.00625 M for 4 h. Same staining. Red filter. Figs 22-31. Eggs of Triturus helveticus Raz. Fig. 32. Morula of Pleurodeles waltlii Michah., treated with glutaraldehyde 0.025 M for 6 h. Same staining. Green filter. Figs 33-36. Eight blastomeres. Egg of Triturus treated with formaldehyde 1 M for 2 h. Figs 37, 38. Eight blastomeres. Egg of Triturus. Formaldehyde 0.1 M for 2 h. Fig. 39. Four blastomeres. Egg of Triturus treated with formaldehyde 0.05 M for 2 h. Fig. 40. Morula of Pleurodeles. Formaldehyde 0.1 M for 10 h. Excepted figs 37, 38 and 39, all the figures at the same magnification as fig. 22.
Gluturaldehyde
andformaldehyde
as antimitotics
Exptl
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239
Res 95 (1975)
240
P. Sentein
nuclei may occur. This explanation presupposes that the lengthening of the spindle is inhibited before destruction of the astral fibres commences, a development which occurs only when these astral fibres begin to expand. (4) Morphology oj’ the blocked mitoses and nuclei. Let us now consider some mor-
phological aspects of the mitoses and nuclei after 0.05 M for 2 h (figs l-7). The types of centrospheres differ according to the stage of mitosis: at metaphase they are granular, not homogeneous (figs I, 2), and ringshaped (fig. 2); between them and the chromosomes appears an achromatic material, incompletely defibrillated (figs 1, 2, 6). In some cases, spindle fibres are still visible and distorted (fig. 6). Chromosomes are in the equatorial region, perpendicular or slightly oblique to the mitotic axis. In some cases the centrospheres appear crescent-shaped in longitudinal section. the achromatic material disappears and the chromosomes are clumped (fig. 5). At telophase, karyomeres are near the centrosphere, but not oriented towards the pole and the single centrosphere is dense, spherical and devoid of astral fibres (fig. 3). This difference between granular and dissociated centrospheres at metaphase, and dense homogeneous centrospheres at telophase, occurs only at strong concentrations and with brief treatment. At telo-prophase the single, densified centrospheres may have a more irregular form (fig. 4). Each of the two nuclei (in fig. 7) in the same blastomere has two centrospheres, but only one is visible on the section. Paradoxically, after 2 h, at a concentration of 0.05 M, the spindle material seems to have been better preserved than at 0.025 M: in the latter case it is completely absent, the centrospheres are nearer each other and Exptl
Cell
Res 95 (1973
more condensed (fig. 1X. to be compared with figs 1, 2 and ’ 6). This better preservation of the achromatic apparatus at strong concentration is an effect of partial fixation. After 0.025 M for 4 or 5 h (figs 8 to 17), at all the phases of mitosis, the fibres disappear completely and the centrospheres are constantly blocked. The latter may be in the process of subdivision (fig. 8). Two centrospheres may be in contact with the nucleus, but not exactly opposite each other (figs 9 and 10, two adjacent sections). In fig. I I, from a morula treated in the same manner, two centrospheres (arrow heads), approximately of the same volume. are at the opposite poles of the nucleus. Chromosomes may be assembled in the equatorial region, but not exactly in the equatorial plane; in figs 12 and 13, two sections from the same blocked mitosis, the centrospheres are on both sides of the chromosome group; that may be considered as a beginning of anaphase. At telophase the karyomeres have the same disposition as the chromosomes at meta-anaphase with regard to the centrospheres: they are not exactly in the equatorial plane (figs 14 and 15, two adjacent sections of the same mitosis). In the prophase of fig. 16 the small nucleus contains fine threads and chromosomes; the two blocked centrospheres are remote from it. Fig. 17 shows a metaphase from a two blastomeres egg treated with 0.025 M concentration for 4 h: the chromosomes are thickened and clumped, but with a shorter treatment of the same solution (2 h, egg treated at eight blastomeres) they are thinner and may be easily distinguished, although not of a normal shape (fig. 18). The same applies for an egg at two blastomeres after 2 h of treatment (fig. 19). The three metaphases of figs 17, 18 and 19 are good examples of the quinoline-like blocked
Glutaraldehyde
mitoses, each with two spherical, densified centrospheres, strongly stained by methyl blue on both sides of the chromosomes. They are also intensely stained by pyronin with the method of Unna (fig. 22), but not if previously treated with ribonuclease. This presence of RNA must be related to the great number of ribosomes observed with the electron microscope in the centrospheres blocked by quinoline (Sentein & Ates (1974), unpublished). Probably the same applies for glutaraldehyde. With the same treatment of a two blastomeres egg for 2 h, the two centrospheres at telophase are of the same type (figs 20 and 21, two adjacent sections of the same telophase): they are spherical, equal to or longer than the centrospheres at metaphase of figs 17, 18, 19. Note that the karyomeres of the left pole (fig. 20) are beyond it; in this case they have overshot the mark. There is only one centrosphere for a group of karyomeres, because the blocking has occurred before its division. From these observations on the action of 0.05 M and 0.025 M concentrations we may note that centrospheres blocked at metaphase may have two types of structure, granular or condensed, while at telophase, telo-prophase, and prophase, they are always condensed and homogeneous. In the latter case they may be formed by a peripheral, intensely stained zone, annular or crescent-shaped, and a less intensely stained central zone. These ‘empty’ centrospheres are more often observed at the lower concentrations (0.0125 M and 0.00625 M). At 0.003125 M, centrospheres may or may not be blocked. The same observations were made with the use of quinoline [14, 15, 161. After 0.0125 M for 6 h, the centrospheres are always blocked and often the nuclei are upper in the animal hemisphere. They begin
andJbrmaldehyde
US rrntimitotics
241
towards the cortex a motion which is less accentuated than with the antimitotics of the colchicine type. In figs 23, 24 and 25 the single centrosphere is near the end of the nucleus, and not in the middle of one of its sides (fig. 23). In figs 26 and 27 (two adjacent sections) the telo-prophasic polyploid nucleus results from the fusion of two groups of karyomeres (as in figs 14 and 15). Consequently the centrospheres are on the same side (arrow head) and not at the opposite poles of the nucleus, as in fig. 1I. The concentration 0.003125 M is the threshold where the fibrillae reappear and the blocking is not constant. A telo-prophasic nucleus from this experiment is shown in fig. 30: some short fibres are preserved around the blocked centrosphere. The action of all the concentrations used was particularly interesting at the time of amphimixy. The centrospheres were always completely blocked, even at the lowest concentrations; astral fibrillae were never formed: this stage is the most sensitive to defibrillation, perhaps because it corresponds to a lengthened telo-prophase. Here also the blocking may occur before or after the division of the centrosphere: in figs 28 and 29 (two adjacent sections of the same amphimixy after 0.05 M for 2 h, stained with methyl green-pyronin) there are two blocked centrospheres at the junction of the pronuclei; opposite to the normal amphimixy, they are not joined by centrodesmotic fibres. But in other cases and especially in fig. 31 (0.00625 M for 4 h) only one centrosphere exists. Similar observations were made with quinoline. The important conclusion of this fact is that under normal conditions there is one division of the centrosphere at the time of amphimixy, that is to say, a separation of the centrioles which are included into it and not a reunion of two centrioles, which might Exprl
Cd
Res 95 (1975)
242
P. Sctitcin
be brought, the one by the spermatozoon and the other by the oocyte. Action of‘gluturuldehyde on the diastemtr The segmentation mitoses are characterized by a special structure, similar to the phragmoplast of the plant cell, which was named diastema; it is fibrillar and situated half-way between the two telophasic nuclei. With glutaraldehyde, some two blastomere eggs may contain four teloprophasic nuclei; between two of them a fibrillar diastema may persist, which indicates the plane which would be normally occupied by the furrow. However, it does not become strongly hyperfibrillar, as under the action of carboxylic acids [13, 191. (5) The action on eggs oj’ Pleurodeles and Bufo. The action of glutaraldehyde on the eggs of Pleurodeles, other species of Triturus and Bufb, does not differ from that on the egg of Triturus helveticus Raz. For instance, from a morula of Pleurodeles waltlii Michah., fig. 32 shows a telo-prophasic nucleus asymmetrically pinched between the two blocked centrospheres. Formaldehyde
Formaldehyde is used as a fixative at 20% of a 37 % commercial solution (2.46 M). The molar concentration has the same effect and, as we shall see, this effect remains partial with 0. I M. The antimitotic effect is observed at 0.1, 0.05,0.02, and 0.025 M (2 h) but 0.0125 M is inactive. The active concentrations being to be toxic after 2 h, sooner than with glutaraldehyde, and produce cytolysis. The formaldehyde has a narrower scale of utilization than glutaraldehyde and the fixation effect overlays the antimitotic one. 2.46 M and M concentrations of formaldehyde give mitotic figures no different in Expti
Cell Res 95 (1975)
size than the normal ones; the achromatic material, clearly delineated from hyaloplasm, is normal in quantity. but the astral and spindle fibres are not preserved (figs 33 to 36). If we compare eggs fixed with 2.46 M formaldehyde solution and post-fixed with Moricard fluid [7], with eggs of the same type fixed from the beginning by Moricard only, we observe that the latter have well preserved spindle and astral fibres, whereas the former have no fibres at all and that the equatorial part of the spindle is not fixed, while in the eggs only fixed by Moricard. this part is formed by conspicuous, well stained fibres. If the sections of eggs fixed with formaldehyde do not show well preserved spindle fibres, it is not because they were not stabilized and subsequently altered by technical manipulations, for instance by alcohols or toluene, but because they are actively destroyed by formaldehyde. Another paradox is that at strong fixative concentration (2.46 M) the centrospheres are granular at metaphase (figs 33-35), while they are homogeneous and more condensed in the eggs of the same stage with 0.1 M after 2 h (figs 37-38); in the latter case the action is mostly antimitotic. For glutaraldehyde, the centrospheres were granular at 0.05 M after 2 h (figs 1, 2, 6) and homogeneous at 0.025 M after the same time, but in the latter case spindle fibres were more destroyed than with the corresponding concentration of formaldehyde (0.1 M for 2 h) and the centrospheres were nearer each other. To explain such an observation, one must think that with formaldehyde at antimitotic concentrations the fixation effect persists longer than with glutaraldehyde. In the particular case of fig. 39, beyond the two blocked centrospheres (arrowheads), secondary poles seem to be formed
Glutaraldehyde
andformaldehyde
as antimitotics
243
PIommL c!ia MIC--suBuL~ITs
depolymerization polymerization
not prevented, prevented at all
levels
l
l.
0 l l oQJlNoLIl+LIKE
-893
progressive microtubules, thereafter
-
birding
to subunits
suEsTz4~cE
m
disappearance of dense bodies and centrospheres .
depolymerization not prevented, poiymerieation prevented at the level of microtubules
c@ 8 00 bin3.ngofsubunit.s or of mre cxxnplex structures between them Probably structures Fig. 41. Possible mechanism types of spindle inhibitors.
a direct wliich
transformtion may be added
of the action
to
The linking of molecules preserves or perhaps strengthens the storage or packaging structures (dense k&ies) of link&l s&units those previously
into formed.
storage
or
packagi-
for the two
Exptl
Cell Res 95 (1975)
(arrows), where normal poles would take place. We explain this fact by a tendency to form again new poles at the limit of the ‘peripheral network’ of the normal metaphase, as we have seen with selenium dioxide [ 1 I] (figs 8, 9). The action of formaldehyde 0. I M on eggs of Pleum<’.s \jxtltlii Michah., is absolutely the same as that on Triturrrs eggs (fig. 40).
At the lower active concentrations (0.02 and 0.025 M) mitoses are more completely blocked in the animal than in the vegetawhere normal bipolar tive blastomeres, mitoses may be observed. There is a grddient brought to light by the antimitotic activity of formaldehyde. A similar gradient was observed in the action of organomercurials (Sentein, unpublished). At 0.025 M after 2 h, centrospheres are completely blocked in the first mitosis of undivided eggs, which were treated before the first cleavage or before the second; on the contrary, the polarity is absolutely normal when treatment begins at four blastomeres or later. Mitosis seems to be protected where and when differentiation begins. DISCUSSION The difference between d-mitosis (quinoline) and c-mitosis (colchicine, vincaleukoblastine, podophyllin) was primarily explained as an effect of fixation by quinoline [20, 211. But even if such an effect exists at higher concentrations, it cannot explain the immobilization of chromosomes at the equator at lower concentrations, since these chromosomes may be transformed into karyomeres without changing place. The only possible explanation is the absence of fibres (microtubules). We have found three additional differences: with colchicine and colcemid the segmentation Exprl
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mitoses are not immediately ,~rrestecl. hut always after at least one mitotic cycle. which is absolutely normal. Secondly. the centrospheres disappear, at least at the light microscope level [4]. Thirdly. blocked mitoses and nuclei go up towards the cortex and/or the animal pole. The same is true of vincaleukoblastin [IO]. On the other hand, the action of quinoline, glutaraldehyde and formaldehyde is immediate and may appear at the first cycle after treatment; mitosis and cleavage are simultaneously arrested and two metaphases are never seen in one blastomere; secondly, these substances produce the ‘blocking’ of centrospheres. There are no conspicuous centrospheres outside the segmentation mitoses. In the normal second segmentation mitosis they reach I7 pm in diameter at prometaphase. In 8 blastomere eggs. blocked centrospheres at metaphase are smaller than the normal ones, 8 instead of I I pm. but the diameter of blocked centrospheres at telophase and telo-prophase may be greater than in normal ones, since they do not dissociate, as in normal conditions: consequently they are roughly similar at metaphase and at telo-prophase. Similar observations are easily explained if we consider that astral regression is the principal source of subunits which furnish the storage structures of the centrospheres. Normally the so-packaged material is utilized at the end of mitosis for astral development. But here, the latter is inhibited and the precursor material remains around the centriole. other material (riboSimultaneously somes and glycogen granules. vesicles, mitochondria and other organelles) may be retained in the centrospheres and remain accumulated therein. Their volume depends on that accumulation. The presence of ARN (pyronin staining suppressed by
Glutaraidehyde RNase) and glycogen (PAS positive) into centrospheres, blocked or not, is evident. The blocked centrospheres without any spindle or astral fibres may be observed not only with quinoline, glutaraldehyde and formaldehyde, but also: (u) with some alkylating agents: nitrogen mustard l/l 000 after 6 h [2, 31 and ethyleneimine-quinones [6]; in the first case no chromosome breakage appears when mitosis is blocked; (6) with organo-mercurials and N-ethylmaleimide, two substances which bind to -SH groups of the proteins; (c) with phenol M/l 000 ([5] figs 1, 2) and amphetamine sulfate M/800 ([7,8] figures l-10,37). The centrospheres may be blocked in the presence of spindle fibres, but always without astral fibres by some carboxylic acids [12, 131. In some cases the tips of the spindle are narrowed and often bent back or coiled ([19] figures 3, 12,35, 39,41, 54). In summary, blocked centrospheres may be observed with a more or less abnormal spindle, but never when spindle and asters persist simultaneously. We must therefore attribute the greatest importance to the construction of asters in order to explain the normal telophasic reduction of centrospheres, and the inhibition of asters to explain their blocking. The nature of this phenomenon will only be understood when an extensive study of the ultrastructure of blocked centrospheres is completed. This study is most advanced for quinoline (Sentein & Ates, unpublished) and it is evident that the blocked telophasic centrospheres include many dense and striated bodies, accumulated at a phase when they are normally absent, and many ribosomes and glycogen granules. The first few results we have obtained with glutaraldehyde (Sentein & Ates, unpublished), are of the same type. The relations between fixation effect and
and formaldehyde
as antimitotics
245
antimitotic action can be explained: in the first case all the cellular structural proteins are linked to many fixative molecules; in the second case only the heterodimeres of tubulin are linked to antimitotic molecules: it is not surprising that more molecules are needed in the first case. But it is evident from our observations that the fixation effect may cloud the consequences of the antimitotic action (though not completely), with formaldehyde. Glutaraldehyde is a better fixative than formaldehyde and exerts its antimitotic action over a larger scale of concentrations. Moreover the pure antimitotic action is observed for the latter at the lower active concentration (0.025 M) and the total disappearance of spindle fibres only in some cases (e.g. fig. 39). On the other hand, even with glutaraldehyde, not all the fibres of the achromatic apparatus in the cleaving egg are as well conserved as with the Moricard fixative. Our observations may be brought nearer to the 50 % reduction of spindle birefringence in the oocytes of Pectinaria by glutaraldehyde, as by colcemid [I]. Acetic acid, which is often associated with formaldehyde in fixative mixtures, namely in Moricard fluid, is known to preserve the spindle fibres very well, because its antimitotic action is different from that of formaldehyde: we have observed that it dissociates the spindle and astral fibres, but usually does not destroy them (Sentein, unpublished). The association of the two substances in suitable concentrations may correct the disadvantage of each of them and give a good compromise for fixation, as observed by all cytologists. Although glutaraldehyde is a better fixative than formaldehyde, its action on the cytoplasm is of the same kind and it is doubtful whether it preserves all the microExprlCcl/
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246
P.
Soltc~itr
tubular structures. Comparison at the light microscope level of half thick sections fixed by glutaraldehyde, with sections fixed by Moricard fluid, is meaningful in this respect. One possible explanation of the differences between the action of colchicine-like and quinoline-like substances is given in fig. 41. In the normal segmentation mitosis, microtubule subunits are packaged into complex, finely fibrillar structures, which constitute dense bodies and probably striated bodies. One molecule of colchicine binds only to one heterodimere of tubulin: the polymerization of microtubules and the formation of storage structures are then simultaneously prevented. On the other hand, to explain the persistence and accumulation of these structures in the centrospheres blocked by quinoline we may suppose a binding of two or several microtubule subunits between them by one molecule of quinoline; these structures would then be stabilized and consequently the polymerization of microtubules would be prevented, as also the breaking up of storage structures. In our first electron microscope observations, glutaraldehyde seems to have had the same effects as quinoline, accumulating dense bodies in the blocked centrospheres (Sentein & Ates, unpub-
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lished). The same explanation may be proposed to apply to both substances. The fixation must be considered an experimental procedure. as the antimitotic action, and the molecular aspects of these two actions, must be studied in the same way. We acknowledge the technical assistance of Huguette Niel. This work was supported by grants from the CNRS, the Fondation pour la recherche medicale francaise, and by a contract from INSERM (no. 72-l-012-1).
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. IO. II. 12. 13. 14. 15. 16. 17. IS. 19.
Inoue, S & Sato. H, J gen physiol 50 (1967) 259. Sentein, P. Compt rend sot biol 149 (1955) 167. Compt rend assoc anat 42 (1955) 1238. Arch anat microsc 45 (1956) 99. Acta anat 34 (1958) 201. Colloq int centre natl rech sci 88 (1960) 143. Chromosoma I3 (1962) 67. Acta anat 49 (1962) 297. Compt rend acad sci 256 (1963) 4759 Chromosoma IS (1964) 416. Ibid 17 (1965) 336. Compt rend acad sci 267 (1968) 1895. Chromosoma 24 (1968) 67. Bull assoc anat 55 (1970) 973. Chromosoma 32 (1970) 97. Compt rend sot biol 164 (1970) 1562. Bull assoc anat 55 (1970) 973. Ibid 56 (1971) 712. Sentein, P & Vannereau, H, Chromosoma 40 (1973) I. 20. Tjio, J H & Levan, A, Anal estac exp aula dei 2 (1950) 21. 21. Lunds Univ arsskr p. I. (1954). Received Revised
January version
3 I, 1975 received April
I, 1975