From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
DNA
Metabolism
Guinea-Pig
and
Development
Megakaryocytes
: A
Autoradiographic
and
By
N
UMEROUS
to the
final
ploidy
by losing
finally
by
thin
section
tion”
and
that
and
have
and
specialize
level
of 8N,
technique
DNA
pigs
cent
weighing
osmium
Short
fixation
These
were
the
ing
on
in
thin
(about
taken
to
Cm.
karyocyte
0.1
have
the
N.
by
jEAN-\IICHEL
the
(IC
‘ploidizaIt
found
was
ploidization
after
an
phase,
average
(PROF.
bone
series
of
the
that
were
for
in
alternate
many
loss
avoided
thick
into
to
with
stainand
(about
10
trans-
a diffuse
dehydrated
2.5
2 ),
might
4
of
because,
1 and
megakaryocytes
megakaryocyte
in
minutes.
materials.18
nonspecific
( Figs.
fixed
seven
it introduces
marrow
were
ultramicrotome
so
7.0
was
Feulgen
method,
the
blocks
of
into
marrow
p11
be
pairs
and
care
being
studied
of
in
sections,
re meats
of
material,
by
the
DNA
containing
Feulgen
about
method.1”
96
Nonspecific
per
cent
staining
of
by
the
mega-
the
Schiff
Hdpital
de Bic#{233}tre, 94-Le
Kremiin-Bic#{234}tre,
Unicersity
of Calif ornia,
Berkeley,
Calif.
accepted
for publication
August
14, 1969.
tile
Soci#{233}t#{233}
Fran#{231}aise (lH#{233}matologie,
December
18, 1967,
Cellulaire,
Laboratory,
23, 1969;
Meeting
of
tile
I., April
a grant
18,
Federation
tile
M.D.:
VAN
of
Associated
Societies
of
Experimental
Biology,
1969.
from
Address:
II.
shown
LKB
each
Mea.s’u
of
Present
of
cell.
Pathologic
PAULUS,
Scientifique.
cut
an
material
stained
Meeting
City,
of
been
osmium-fixed
Donner
tile
then
with
metric
of
May
at
blocks
phosphate
in the
fixation,
divided
of
were
tile
Supported
Liege,
lo:s
thus
size
.snbnlitted
tile
were
sections
17l.StitUt
tile
Atlantic
Medicale.s,
The
NI
Small
the
photo
and
used.
adequate
. )
nuclei,
at
and
and
METHODS
be
sections
Presented
and
during
the
had
stopped
to
the
pectro
thick
First
AND
were
in
osmium
cutting
on
From
began
process
matures
between
level.
in the present
study
to try to minimize
associated
with this fixative.
Clutaraldehyde
in
minimize
depending
France,
relationship
ultrastructural
at
epon.
0.08
This
The
cytoplasm
platelet
antigens,1
the alternate
thick
buffer
times
preferred
eml)ed(led
298
300
Schiff
reagent
used
subsequently
of hone
marrow
cells.
After
Micros
and
study,
the
the
organelles
when
this
tetroxide
(NSLT)
totalit.
cell
that
leads
of Sections
Guinea
mission
relationships
syntheses.
Preparation
ier
of the
to explore
MATERIALS
1
between
Light
microscopy
studies
show
first, a series
of DNA
syntheses
granules10’
the present
maturation
the synthesis
of specific
increased
considerably
of three
made
or 64N7#{176};secondly,
32N
used
was
cytoplasmic
Study
PAULUS
been
16N,
in
Ultrastructural,
Cytophotometric
proliferation.16
in two steps:
its basophilia,
acquiring
liberating
platelets.17
In
Organelles
Combined
JEAN-MICHEL
ANALYSES
differentiation
megakaryocytes
of
French
Goeerntnent.
Charg#{233} de Recilerches
Institut
COUWENBERCE
(Ic
Medecine,
AND
au
Fonds
Beige
D#{233}partement
Assoc.
PROF.
de
J.
(IC
la
Recherche
et Patilologie
Clinique
Unicer.sity
HuGuEs)
of
Belgium.
BLOOD,
VOL.
35,
No.
3
(MARCH)
1970
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DNA
METABOLISM
AND
THE
DEVELOPMENT
OF
299
ORGANELLES
Fig.
1.-Scheme
m a r r o w
sectioning,
2500
showing
cut
thick
sections.
five
thin
four
For
tions
80
megakaryocyte
into
and
of
clarity,
thin
magnified
proportion
sections
times.
secout of
thick
three
to
about
Fig.
2.-Illustration
of method.
Fig.
2a.Phase
- contrast
micrograph
of
3..
section
of osmium-fixed
karyocyte,
megastained
by
Feulgen
trioles
method.
Ceii(ce) and one demarcation
111 e in b r a Ii e
(dm)
are
conspicuous
(x 5000).
Fig.
2b.-Portion
of
r e c o r d in g of DNA
measurement
made
on
above
megakaryocyte.
Section
of n u c I e u s
was scanned
along
four
adjacent
lines
bered
each
light
one
i-p.
and
4000
ment
at
I
and
was
both
at
x
four),
vicle
transmission
recorded
_______________
iA
%J ‘
#{149}- ‘-
.
I
___________V
5600
A.
Measure-
4000
A
per
mits correction
for
13
per
cent
nonspecific
1 o s s of
transmission
created
b\ osmium
fixation and allows
calculation
of
7.9N
(see
Fig.
ploidv
2a.,
value,
thin
section
showing
megakaryocyte
demarcation
endoplasmic
in
w
membranes
reticulum
it
A
I
I
F
‘
‘F’
-
____________
____________
____________
&
_______________
______
___________
___________
(dm),
5000).
granules,
w
Al
___________
-V V
I
r
____________U
‘
p
_______________
rk
_______________
__________
__________
IvJ
Al
________
__________
________
-
__________
c
--3
2
4
_____________________
___________
h
(x
iI
I’,
___________
_____
maturing
‘.
I&
IIv
__________
__________
Fig.
_______________
___________
-__________
to
‘
I
,I
-__________
AA
sec
1
.,
V
___________
________
of
________________
A
jPI
l#_I
___________
methods).
contiguous
thick
-
5O
TA
_______________
2c.-Electron
micrograph
tion
-
(numto
four
centrioles
(ce),
ribosomes
and
rough
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
300
JEAN-MICHEL
Fig.
PAULUS
3.-Extinction
C U r v e s of
megakary-
nucleus
in
bone
marrow
s m e a r
fixed
with
formol
and
stained
by F e u 1 g e n
method.
B: nucleus
(nu)
and
cytoplasm
(cy)
in a section
of
marrow
following
osmiurn fixation,
epori emocytes.
A:
bedding
and
,
Feulgen
procedure,
omitting
staining
by Schiff
reagent.
C: nucleus
in a
section
of marrow
following
o 5 rn i u m fixation,
epon
embedding
and complete
Feulgen
procedure.
Cytoplasmic curve
was
similar
to B or showed
a small, negligible
extinction,
using arbitrary
units
concentration
of
reagent
was
absorbing
absent
peak
so that
tray
at 560 m. Curves
their shape
might
were plotted
as logarithm
be made
independent
of
material.22
negligible.
or
Waveleagth
However,
osmium
fixation
introduced
a nonspecific
loss
of
both in the nucleus
and the cytoplasm.
Although
this NSLT
in the
past did not prevent
Feulgen
measurements
of DNA,2#{176}it invalidated
uncorrected
scanning
measurements,
which
cannot
be limited
strictly
to the nucleus.
The
NSLT,
highest
at
400
m.
(Fig.
3), is explained
by the light scattering
caused
by osmium-fixed
subcellular
particles.2’
The extinction
spectrum
over the nucleus
was approximately
the sum of the
transmission
NSLT
(NSLT),
and
the
extinction
of
formol-fixed
nuclei
In addition,
the Feulgen
per cent of that measured
extinction
on the
at 560 mi. Since
in
nucleus
both
cytoplasm
and
“baseline”
NSLT
control
extinction
560
at
mj.
for
could
(Fig.
each
be
stained
by
latter
material
the spectrum
3),
the Feulgen
nucleus
measured.
calculated
the
Feulgen
method
Consequently,
according
(Fig.
3).
at 400 mj. was only about
seven
of the NSLT
was almost
the same
negative
cytoplasm
served
as a
to the
the
following
specific
Feulgen
formula
E400
F560
in
which
and
in
E5611 and
k (which
the
E400
varied
absorbing
between
cytoplasm
the
Zeiss
NA
The
0.3. The scanning
total
DNA
of
measured
ments
UMSP1
operated
Labeling
used.
two
(Fig.
et
and
2).
E560
-
measured
four)
k
extinction
is the
Scanning
at
ratio
of
560
ploidy
value
normoblasts,
was
known
NA
1.25
be
respectively,
measured
performed
and
were
up
from
with
a condenser
diaphragm
adding
determined
to
mp.,
NSLT560
was
Ultrafluar
objective
100 x,
the diameter
of the measuring
megakaryocyte
was
calculated
by
the
400
microspectrophotometry
with an
interval
and
each
and
NSLT4#{216}0 to
the
similar
1
jz.#{176}
amounts
measure-
diploid.23
Studies
of DNA-synthesizing
al.24
Blocks
#{176}Inpreliminary
difference
the
for each section,
and
made on orthochromatic
Autoradiographic
Rubini
represent
=
in mean
of
megakaryocytes
guinea-pig
marrow
experiments
conducted
nuclear
extinction
was
was
were
by
found
carried
incubated
out
in
for
one
the multiple
plug
when
diaphragms
vitro
method,
of 0.32
described
as
hour
at
37#{176}C
no
j.
by
in
significant
or 1
were
a
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DNA
METABOLISM
AND
THE
DEVELOPMENT
OF
301
ORGANELLES
Cl)
a)
4-’
>
Co
CO
a)
E
Co
4-’
0
9-
0
4-’
C
a)
C.)
a)
a-
Ploidy
Fig.
ploidy
values
of
guinea-pig
megakaryocytes,
using
above-
technique.
labeling
solution
3H),
specific
serum,
buffer
buffer
consisting
of
activity
thick
and
on a glass
placed
weeks.
fixed
for
six
contrast
Electron
thin
minutes
of
fetal
(CEN,
calf
serum
Mol,
in
cent
per
20
jCi.
After
of
thymidine
washing
glutaraldehyde
cent
osmium
( methyl-
with
solution
tetroxide
fetal
in
in
calf
phosphate
phosphate
epon.
sections
in
and
Belgium).
fixed with a six per
for 40 mm.
in one
slide,
covered
the slides
Subsequently,
phase
1 ml.
15C/mM.
the blocks
were then
at pH 7.0, postfixed
and finally
embedded
Alternate
and
of
4.-Histogram
described
were
cut,
as
with Kodak
were
developed
thiosulfate
fixer.
described
AR
10
for
above.
stripping
six minutes
Finally,
they
The
film
thick
and
in Kodak
were
mounted
sections
were
exposed
for
D170
developer
and
five
examined
in
microscopy.
Microscopy
The thin sections
were stained
by uranyl
acetate
and lead citrate,25
and
examined
tinder
a Siemens
or a Hitachi
electron
microscope,
operated
at 80 or 75 KY, to assess
the
stage
of the cell cytoplasmic
maturation.
Low magnification
photographs
of the thick sections
were
used to correlate
the microspectrophotometric
or autoradiographic
findings
on each megakaryocyte
with
its
electron
microscopic
characteristics
(Figs.
2 and
5).
RESULTS
Relationship
between
Ultrastructure
DNA
megakaryocytes
of
megakaryocytes”#{176}
were
mitotic
cells.
Megakaryocytes
#{176}Immature
and
not to
immature
was
Bessis’
basophile
than
here,
those
and
ability
during
correspond
since
to
which
it was
described
granular
the
by analysis
incorporating
megakaryocytes,27
used
to cells younger
to
studied
megakaryocytes
basophile
synthesizing
felt
that
in this
megakaryocytes.
and
ploidization
of both
thymidine
megakaryoblasts
are
cytoplasmic
non-DNA
maturation
phase.
DNA
had,
in
“Immature
synthesizing
in addition
Bessis’
synthesizing
and
to the
nomenclature,26
cells.’5
The
term
the name megakaryoblast
should
be reserved
study.
Maturing
megakaryocytes
corresponded
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
302
JEAN-MICHEL
Fig.
PAIJLUS
5.-Immature
synthe-
megakaryocyte
sizing
DNA.
radiogram
tion
of
of
bated
one
contrast
5b:
Colgi
cell
5c:
presumably
ce.
hyde
6a,
6b),
with
and
the
partially
rest
of
cells
mature
the
a central
This
assumes
possibly
also
in size.
ribosomes
and
in
the
(2)
(Fig.
cytoplasmic
aspect
shape
of
Although
5,
6),
number
(1)
that
a round
immaturity
was
well developed
a small
osmium
5) or ring
area
suggested
In sections
cut through
the
youngest
cells,28
the central
polyribosomes,
(Figs.
the
U shaped
of
which
the
mass
interpenetrated
nucleus
zone
the
from
and
the
the
specific
formed
in most
of the immature
megakaryocytes.
The
zone contained
a large
Golgi
apparatus,
often
extending
5, 6),
and
known
to be
synthesizing
the
early
the
ma(Figs.
by
(Fig.
6c)
was much
abundance
of
rather
diffuse
organelles
pattern
already
plasmic
(Figs.
In
shaped
communicated
nucleus
of im-
nuclear
cytoplasm
fixation
top of the
cytoplasmic
evident
nucleoli,
cen-
Glutaralde-
7000).
characteristics.
distinctive
either
encircled
extensions.
reduced
two
was
cytoplasm.
roughly
cytoplasmic
and
ratio,
nucleus
micro-
near
triole
the
Ar-
to
originating
sections,
to
zone
of
showing
points
t u b u 1e
of
sec-
formation.
row
nuclear-cvtoplasmic
Thin
corresponding
same
granule
joritv
la-
fuchsin
phase
1000).
5a.
high
in
(u)
and
megakaryo-
Basic
staining,
(x
hour
showing
(L)
cytes.
tion
see-
incu-
thymidine
unlabeled
beled
(x
thick
marrow
for
aJ1
Auto-
5a:
central
into the
granules
were
cytoouter
(Fig.
5; Ref. 29).
Microtubules
(Fig.
5) and,
in other
cytoplasm,
accompanied
appeared
to originate
in the vicinity
of the centrioles
pictures
they
were
visible
at the periphery
of the
by myofibrils.
Demarcation
membranes
were
also
found
In addition
in
immature
cells.
to these
organelles,
immature
megakaryo-
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DNA
METABOLISM
Fig.
AND
6.-Sections
THE
through
DEVELOPMENT
immature
OF
303
ORGANELLES
A,
megakaryocytes.
B: Showing
central
cyto-
plasmic
zone partially
encircled
b nuclear
lobes. Autoradiography
of adjacent
thick
section
demonstrated
thvmidine
incorporation.
Glutarakiehyde
and osmium
fixation
x approximately
3500. C: Section
through
top
of nucleus.
Ploidy
value
is indicated.
Osmium
fixation
(x 7300).
cytes
(Fig.
contained
many
mitochondria
and
5). 17.5 per cent
of megakaryocytes
described
above.
In mitotic
megakaryocytes,
microtubules
were
Ultrastructure
megakaryocytes
after
represented
the majority
Although
in some
sections,
encircled
the
sive
by
nucleus
Cytoplasmic
granule
synthesis,
(Fig.
of rough
endoplasmic
reticulum
the cytological
characteristics
demarcation
7),
membranes
and
visible.28
megakaryocytes
of the series.
uration.
had
granules,
similarly
of
profiles
was
development
formation
still
visible,
in these
of large
the
ploidization
(67.5%)
the central
it appeared
cells
amounts
phase.
Maturing
of the recognizable
cytoplasmic
zone
less
involves,
frequently
in
addition
of demarcation
elements
partially
with
mat-
to extenmembranes
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304
JEAN-MICHEL
PAULU5
:4b
Fig.
7.-Immature
megakaryocyte
paratus
and
early
(x
620(fl.
Insert:
(Figs.
8. 9),
Since
the
which
cytoplasm,
the
has
an
outer
zone
becomes
aspect
came
more
and
far,
and
megakaryocytes
and
attached
to the
bubbling
territories
ap-
(Fig.
intermediate
concentric
cells
With
while
formation
of the
The
(Fig.
progressive
disappeared,
9c).
part
zones.
of younger
membrane
10,
In
some
cases
remnants
of
between
The
values
ploidy
12.5
per
cent,
32N
and
the
of
inner
9a).
The
maturation,
mitochondria
be-
and
be-
zonation
and
peaks
three
No
the
cells,
Newly
bore
with
total
were
into
the
Cytoplasmic
4N
was
peripheral
included
in
platelets.
11),
prethis
group
Maturation
at 8N,
20
megakaryocyte
coefficient
(Figs.
megakaryocytes.
of frequency
classes
still
vessel
a hypertrophied
also
a greatly
platelets,
formed
protruding
cells,
of the
reached
group,
seen
platelets
Polyploidy
these
pycnotic
10).
absence
of this organelle
in circulating
by a narrow
band
of cytoplasm
(Fig.
cent
respectively.
16N
be
released
near
generally
(Fig.
thrombopoietic
15 per
Relationship
proportion
zone
could
the
with the
surrounded
represented
The
were
marginal
megakaryocyte,
zone contrasting
Pycnotic
nuclei
sumably
three
microtubules.
thus
numerous,
platelet
to the
into
Golgi
conspicuous.
enlarged
which
future
to that
and
present
less
Thrombopoietic
9d).
divided
showing
Electron
micrograph
section
(x 7470).
thick
is restricted
similar
myofibrils
ribosomes
the
mitosis,
fixation.
adjacent
delineate
latter
of
osmium
of
of membranes
immature
and
smaller
ana-telophase
picture
eventually
contains
came
in
Glutaraldehyde
phase-contrast
formation
zone
nucleoli
granules.
of variation
per
16N
cent,
could
and
67.5
be
between
32N
per
recognized.
individual
(Fig.
cent
4).
and
In
the
mega-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
DNA
METABOLISM
Fig.
8.-Similar
AND
ploidy
value.
karyocyte
DNA
content
Figure
each
cell
cells).
12 shows
the state
DEVELOPMENT
cytoplasmic
of different
immature
THE
organelle
Osmium
was
fixation
9
and
OF
development
(x
5700).
10
per
a diagram
of megakaryocyte
of cytoplasmic
maturation
305
ORGANELLES
in
cent,
maturing
respectively
megakaryocytes
(excluding
maturation
versus
ploidy.
and the degree
of ploidy
For
have
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
306
JEAN-MICHEL
PAULUS
Fig.
9.-Successive
stages
of
cytoplasmic
specialization
in
maturing
one
show
ferent
ploidy
newly
platelets
protruding
Black arrows
indicate
limits.
Lobes
mium
3100).
top
of
vessel
of
akaryocyte
zone.
vessel.
in
figure
labeled
bling
trophied
(pI)
into
meg-
nucleus
nu.
(bub)
(See
Note
bubof hyperperipheral
Fig.
fixation
9d).
Os(x
Platethrough
wall
(v)
visible
sides
of
let
(see
Fig.
10).
(a)
(c)
megakaryocyte
levels.
formed
both
9700).
c
of
clif-
on
fixation:
8100),
five
at
protruding
mium
and
b,
iii e ni
(dm)
vessel
10.-Thrombo-
a,
development
branes
let
Fig.
and
(d)
demarcation
poietic
c)
thrombopoietic
megakaryocyte.
d
three
b,
(a,
(b)
(x
(x
29600),
plate-
Os(x
74:300),
(d)
(x
-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
DNA
METABOLISM
rI
AND
THE
DEVELOPMENT
OF
307
ORGANELLES
‘#{149}
‘
#{149}“
#{149}#{149}i,Lt
r
-
Fig.
1 1.-Pycnotic
megakaryocyte
vs.
(x
been
determined.
ploidv
had
levels
Immature
8N
conipleted
levels
SN,
to
a
or 32N.
the
other
was
cells,
ploidy
levels
be
illustration
have
a nearly
a
error
thrombopoietic
three
could
32N
of thrombopoietic
had
been
made
photometric
able
to
and
As an
which
16N,
Study
analyses
cells,
Maturing
ploidization
16N
megakarvocytes
is
23N.
were
found
of
the
Figure
submicroscopic
at
which
three
ploidy
8 shows
two
appearance.
One
cell.
relatively
(Figs.
each
observation,
identical
12,600).
DNA,
at
of this
fixation
megakaryocytes
encountered
megakaryocytes
on this stage
it was
synthesize
thrombopoietic
nude-
Osmium
was
before.17
larger
clear
that
on
of particular
Despite
the
the
condensed,
thrombopoiesis
interest
fact
since
few
that
micro-
pycnotic
could
be
nuclei
observed
of
at all
10, 11, 12).
DIsCussIoN
Method
The
The
used
study
technique
to
shows
cellular
ting
The
that
substance,
nuclei
16N
and
cent,
67.5 per
similar
values
alternate
and
thin
sectioning
DNA
in relation
to the
cell’s
of quantitative
cytochemistry
procedure
is demonstrated
by the
of guinea-pigs
32N
thick
has
been
electron
microscopy
and
autoradiography.30’3’
it also makes
it possible
to measure
the relative
i.e.,
the combination
validity
of this
yocyte
8N,
of
combine
could
(Fig.
4);
cent
and
obtained
12.5
by
the
be
grouped
proportion
per cent,
conventional
ultrastructure,
thus
permit-
and electron
microscopy.
following
facts:
Megakar-
in three
of these
previously
The
present
amount
of a
ploidy
three
classes,
classes
respectively,
to be compared
microspectrophotometry
namely
was
20
per
with
very
in guinea-
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
308
JEAN-MICHEL
32N
I.
.-
16N5
I
J:..
I
i-
>-
PAULUS
____S
S
:
Fig.
:
12.-Diagram
ploidy
I
in
developing
Each
megakaryocytes.
:
8N
of
values
represents
one
cell.
for explanation
point
See
text
of arrows.
4N
Immature
pigs’2
and
DNA
content
errors,
Maturing
rats’3;
since
very
small.32
of
error
Pattern
The
among
was
dispersion
the
ploidy
present
study
light
to
tion
of
specific
power
of the
early
shows
granules.
several
ing
zone
not
most
this
only
membranes,
phase.
the
thrombocytic
are
class
method
way
between
modal
of
of variation
mainly
of
technical
is
probably
value
is sufficient
shows
neither
that
strictly
two
to exclude
parallel
ultrastructural
analyses
immature
megakaryocytes
in
discrepancy
are
to
microscope,
Golgi
the
sign
series
but
an
also
incorporate
in
the
early
cytoplasm
of cytoplasmic
Odell
not
consecutive
and
in partial
detect
some
in
the
particular
and
granules.
5, 6).
maturation
myofibrils
et al. have
into
5,
forma7).
superior
The
resolving
appear
of
the
in sections,
zone contain-
central
Granule
in immature
also
shown
agreement
early
localization
The
(Figs.
of
processes.
(Figs.
only
the
conceptions
development
the nucleus
appears
solid,
encircle
a central
cytoplasmic
and
outer
microtubules
In addition,
found
in imprints
partially
apparatus
with
studies,
be
and
nor
autoradiography
organelles
opposite
ploidization
microscopy
detectable
zation
the
of the
a middle
and
Whereas
lobes which
cation
coefficient
consists
of Kinosita,33
communicates
the
proposes
electron
of the
the
Maturation
organelles
studies
classes,
variation
around
precision
maturation
the
32N
This
class.
Megakaryocyte
with
and
cent.
biological
of
for
16N
10 per
Therefore,
one
cytoplasmic
reasons
the
9 or
of megakaryocyte
Contrary
Thrombopoietic
during
that
all recognizable
chondroitin
sulfate,34’35
it
cytoplasmic
formation
cells;
is
demar-
the
ploidi-
cells
the
acid
of
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
DNA
METABOLISM
AND
DEVELOPMENT
THE
mucopolysaccharide
presumably
synthesize
DNA.
much
less
frequently
were
made
in light
conclusion
common
phase
tion
This
and
8N
can be inferred
thrombopoietic
were
and
found
32N
the
average,
3
±
is marked
(1
for
the
by several
latter
become
granules
and
apparent
and
karyocyte
that
reaches
)
12
Similar
.
series.
of platelet
changes.
which
At this
pro-
The
nucleus
best
gradually
stage
observed
ceases
as
cytoratio.
occurs
it is possible
is thrombosthenin,
present
formation
after
bears
the
some
Granulocytes
is initiated
ploidization
resemblance
as well
in immature
phase.
to
This
in
of mega-
specialization
as megakaryocytes
element,
the
metabolic
activities
of
begin
of which
have
mega-
pattern
the
other
synthesizing
ules in the Golgi
apparatus
during
the phase
of genome
multiplication.
series,
nuclear
segmentation
accompanies
the end of this phase.
both erythrocytic
and megakaryocytic
series,
after nuclear
pycnosis,
enucleate
findings
size remains
constant’5
the nuclear-cytoplasmic
membranes
and
granules
microscopy.9’1#{176}
and
duplications.
regulation
cytological
group
Jackson.’#{176} Thus,
the
is that the ploidiza-
) genome
S.D.
measurewhich
16N
mass,
a modification
The Golgi
apparatus
among
organelle
a peak
maturation
marrow
in light
antigens15
of de-
membranes.3#{176}
It is thus
karyocytes
visible
platelet
staining
in the
(Fig.
0.5
encircled
by the nucleus.
The
nuclear
volume
increases,
thereby
decreasing
a massive
synthesis
of demarcation
the
blue
from
ploidy
megakaryocytes
mostly
groups17
mechanism
into an irregularly
segmented
but evident
in sections
also.
to demonstrate
alcian
the
by de Leval,#{176}and Odell
and
and electron
microscopy
studies
a possible
of ploidization
is transformed
in imprints,’5
to be
plasmic
Finally
on
suggests
duction.37’38
The end
cells
in the
comprises,
finding
These
microscopy
of light
for
phase
and
309
ORGANELLES
responsible
marcation
membranes.27’35f
The length
of the ploidization
ments
on cells
such
as maturing
never
OF
many
gran-
In both
In addition,
produce
an
similarities.37
SUMMARY
The
thick
and
karyocytes
by
thin
electron
cytophotometric
ization
in
already
showed
f The
It
occurred
origin
of
considered
should
be
smooth
B.M.,
but
cells.
functions
elements.
and
out
White,
that,
as
References
J. C.: Uptake
which
demarcation
organelles
Platelet
membranes
reticulum
a
only
as
muscle
has
calcium
pertinent
of
calcium
began
been
in the
of
controversy,
or
cell
but
membranes,
extrusion
by
megaor
in polyploidGolgi
phase
stopped
S.D.)
genome
a matter
sarcoplasmic
to this
guinea-pig
autoradiography
engaged
reticulum39.37
cell
study
either
apparatus
at the 8N,
duplications.
membranes
were
also present
in the
were
formed
in increasing
aniounts
liberation,
possible
at the 8N, 16N or
cells,
principally
at the 16N level.
endoplasmic
not
to
with
The ploidization
of 3 ± 0.5 (1
DNA-synthesizing
as smooth
pointed
endoplasmic
non
used
Megakaryocytes
formation,
and
demarcation
either
polysaccharides,
troctile
of DNA.
granule
in
was
combined
by the nucleus.
after
an average
myofibrils
megakaryocytes
stage,
been
microscopy,
DNA-synthesizing
non
32N
technique
determination
partially
encircled
16N or 32N level,
Microtubules,
immature
section
and,
pump
problem
platelet
also
such
reticulum
are
relaxing
given
is
is
in
by
factor.
since
these
membrane27
have
derivatives.
a derivative
lined
close
Statland,
Nature
with
of
acid
proximity
B.
to
E.,
223:521,
the
mucocon-
Heagan,
1929.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
310
JEAN-MICHEL
PAULUS
ACKNOWLEDGMENTS
This
M.
work
Bessis,
Corius
has
to
whom
and
B.
assistance
possible
Thorell
of
for
0.
thanks
extend
was
we
experience
whose
Mel
been
the
generous
in
excellent
criticism.
J. Maigne,
help
and
invaluable
We are also very
thanks.
indispensable
their
Pierre,
to
heartfelt
this
study,
We
wish
M. Coumont,
R. de
advice
grateful
and
to
Drs.
finally
to
acknowledge
Postel,
and
of
Dr.
to Mrs. J. BretonG.
C.
Brecher,
H.
the
C.
skillful
Zarka.
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DNA
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and
1969;
AND
Thorell,
THE
B.:
DEVELOPMENT
OF
communi-
au
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31.
1963.
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#{233}lectronique
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Cytochemical
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endocellular
Blood
Grasso,
H.:
B.:
erythroblasts
to the
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Swift,
Thorell,
determination
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E.
microscope
sang
Microspectrophotometric
311
ORGANELLES
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
1970 35: 298-311
DNA Metabolism and Development of Organelles in Guinea-Pig
Megakaryocytes: A Combined Ultrastructural, Autoradiographic and
Cytophotometric Study
JEAN-MICHEL PAULUS
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