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Effect of colchicine on mitosis in the neural tube of the forty-eight hour chick embryo.

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EFE'ECZ' O F CO1,CIIICINE ON MlTOSIS IX THE NEURAI,
TUBE O F THE E'ORTY-EIGHT HOUR CHICK EMBRYO'
T. M. WOODARD, JR. AND SARAH B. ESTES
Department of Biology, Vanderbilt University, iVashville, Tenn.
INTRODUCTION
OIW of t l i ~d f w t s of colchicine upon mitotic nuclei is to produce
inetaphase block. A t adequate concentrations and as time goes 011 more
aud I ~ O I ' C nuclei a r e thus affected, so that in counts the number. of
mitotic iiuclei appears to illcrease. This increase was first interpreted
by many to mean that mitosis had been stimulated. It was then realized
that if all mitosis in progress at a giveii time is arrested at the metaphase ail increase might be the result simply of a n accumulation of
nuclei in this phase (Ludford, ' 3 6 ) .
The latter interpretation is now geiierdly accepted, but even yeceiitly certain results with colchicine on animal material have been
attributed to stiniulatiou. This is the case, for example, in the chick i i i
which PafY ( '39) found that one result of treating 48-hour' chick cnibryos with (.olehicine was to pi-oduee local regions of overgrowth in
the neural tube. These he attributed to mitotic stimulation. It seeins
desirable, therefore, to determine in a quantitative way, if possible,
whether or not mitosis is stimulated by colchicine. This is especially
important in view of the frequency with which colchicine is being used
to arrest mitosis as an experimental method.
MATERIAL AND METHODS
The window tecluiic of introducing substances to the embryo chick
leaves much to be desii*edin speed and in the assurance that the embryo
is uniformly surrounded by the substances introduced. Although we
tried this method and various niodifications of it in preliminary esperiments, it was soon abandoned in favor of the method described
below which we found to be simpler and to give more uniform results.
Eggs were incubated slightly over 48 hours. Each egg was then
opened, and most of the contained albumen was poured into a 100-cc.
beaker containing 10 cc. of a solution of colchicine in a warmed, physiThe senior author is indebted t o the Division of Natural Sciences, Vanderbilt University,
for financial aid in this work.
51
52
T. M. WOODARD, JH. A N D SARAH B. ESTES
ological salt solution. The albumen and colchiciiie solution were then
mixed by agitation and the intact yolk and remaining albumen were
added. A strip of gauze was wrapped around each beaker so a s to
project over the rim, and a glass plate was rested on the edges of the
cloth as a cover. Each beaker with contents was placed in a n incubator
where it remained for some measured time.
Embryos so treated and controls were removed from the yolks in
the usual way. They were fixed in warmed B-15 fluid, dehydrated very
gradually, cleared and imbedded. Sections were prepared and stained
with iron alum hematoxylin. Counts were made of the number of nuclei
in each of the phases of mitosis and of the number of resting nuclei. A
modified Howard micrometer disc ruled into nine squares was used in
counting. Counts were recorded for each micrometer area, and these
were combined at random until a total number of nuclei near 2000 was
reached. These totals were then used as samples in determining the
standard deviations and were further combined for presentation.
Special care was taken to make counts here and there over the neural
tube, since it is known (Derrick, '37) that the mitotic index varies widely
in different regions.
The method described above of maintaining embryos during the experimental period must furnish conditions which are essentially normal
well beyond the duration of the experiment ( 3 hours), for controls
(10 cc. of salt solution plus albumen) were normally developed after
24 hours. Incidentally several embryos were carried successfully well
beyond this period, even up to 2 and 3 days.
Concentrations of colchicine ranging from 0.0078 mg., to 1.0 mg., per
embryo were tried in preliminary experiments. At all of these concentrations effects on mitosis appeared as early as 20 to 30 minutes after
treatment had begun. Since it was not desired to carry these experiments beyond a few hours, that dose was selected (1.0 mg.) which produced complete block of most nuclei within 3 hours. All of the data
here recorded are based upon embryos treated with this amount for.
periods of time ranging from 40 minutes to a maximum of 3 hours.
OBSERVATIONS A N D DISCUSSION
The results of our experiments are shown iit table 1. Considering
the percentage of each phase of mitosis referred to the total number
of iiuclei counted, the data show clearly that there is no increase in the
percentage of any phase except metaphase. If colchicine acted as a
stimulant there would have to be an increase in all phases of mitosis.
The increase in nictaphase can be entirely accounted for in terms of
53
EFFECT OF COLCHICINE O N lllITOSIS
accumulation of arrested cells. Thus, anaphases 2nd telophases have
almost completely disappeared after 40 minutes, while at this time theper cent of prophases is still almost unchanged, indicating that cells
are still coming through prophase to metaphase at the normal rate.
The increase in metaphase during these 40 minutes, from 2.4% to 10.6%,
represents the nuclei which during this time have entered metaphase.
TABLE 1
Effect of 1.0 wig., of colcliicine on mitosis in the neural tube of the 48-holrr chick. The percentage of each phase of ~tittosisis based on the total n z c ~ n b ~ofr nnclei c o w t e d . I n some C U S P S
only prophases were counted an certain samples, so that the s u m of the percentages o f each
p?&uscmay not always eqical the nzitntic index.
TIME
TREAThn
Control
40 minutes
1hour
lthour
2hours
Bhours
NUMBS11
OF
NUCLF,I
COUNTXD
NUMRHR
OF
MITOTIC
NUCLEI
6363
14534
3928
12816
19074
19579
598
1989
627
1670
3240
3977
MITOTIC
INDEX
ST A NDAH n
DSVIATION
9.3
13.6
15.9
r 1.1
13:O
17.1
20.2
-t 3.8
& 3.1
& 4.2
* 1.1
PER CUNT
PKOPHAEE
2.8
2.4
3.0
2.3
0.6
1.1
PER,C E N T P E R C E N T P S K C B N 3
MEFAPHARE
2.4
10.6
8.4
9.2
15.8
20.5
ANAPHASE
TELOPHASE
0.2
0.0
0.0
0.0
0.0
0.0
4.6
0.3
0.0
1.4
0.1
0.0
In view of tile results of these experiments the rcgious of excessive
overgrowth ill the neural tube described by Paff ('39) a i d observed in
our material also must be attributed to some other cause tliaii colr11ic:ine
stimulation. Sauer ( ' 3 5 ) has shown that in the neural tube epithelium
nuclei migrate from the peripheiy into the germinal layer where they
then undergo division, returning then after completing division. Since
colchicine arrests the division at metaphase, it may be that the return
of resting nuclei is also prevented but their migration into the germinal
layer is not. I f this be true, nuclei would then tend to accumulate in
the germinal layer producing the characteristic swellings or regions of
apparent overgrowth.
SUMMARY
1. Chick embryos of 48 hours iiicubatioii were treated with 1.0 mg.,
of colchicine and allowed to continue development for different lengths
of time. Counts were then made of the number of resting and mitotic
nuclei in the neural tube.
2. The mitotic index rises to a maximum of 20.5% in 3 hours. The
percentage of each phase of mitosis, except that of metaphase, eventu-ally declines. The rise in metaphases can be accounted for in terms of
the accumulation of nuclei i n this phase. It is concluded that colchicine
at this concentration acts only in this way and not as a mitotic stimulant.
54
T. M. WOODARD, JB. AND SARAH B. ESTES
LITERATURE CITED
1937 An analysis of the early development of the chick by means of the
mitatic index. J. Morpli., vol. 61, pp. 257-284.
LUDFORD,
R. J. 1936 The actioii of toxic substances upon division of normal aiid malignaiit
cells. Arch. exp. Zellf., vol. 18, pp. 411-441.
PAFF,G. H. 1939 Action of eolehieine upon the forty-eight hour chick embryo. Am. J. Anat.,
V O ~64,
. pp. 331-348.
SAUER,
F. C. 1935 Mitosis in the neural tube. J. Comp. Neur., vol. 62, pp. 377-405.
DERRICK,
G. E.
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colchicine, effect, mitosis, hours, embryo, neural, chick, eighth, forte, tube
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