Download Changes in DNA, RNA, and Protein Synthesis in the

Changes in DNA, RNA, and protein synthesis
in the developing lens
Calvin Hanna
Lens cell DNA, RNA, and protein synthesis in the developing mouse eye were studied with
the use of tritium-labeled thymidine, uridine, and l-leucine and autoradiographic techniques.
In the mouse embryonic lens, epithelial cells undergoing DNA synthesis were found over the
entire anterior lens surface. From birth and until the eyes opened the percentage of epithelial
cells undergoing DNA synthesis rapidly decreased. Later the percentage of epithelial cells
undergoing DNA synthesis teas nearly constant as the germinative zone became localized in
the lens equator region. RNA synthesis occurred in all nucleated cells of the developing lens
from the embryonic stage until the eyelids opened. With lens maturity the SH uridine was
incorporated into the RNA of only the more superficial cells. A similar pattern of tritium incorporation was seen with SH l-leucine.
taining 3H-thymidine. The lens capsule
with epithelial cells attached was removed
and placed on a glass slide (flat mount)
and covered with photographic emulsion.
An analysis of the resulting autoradiogram
revealed tritium-labeled epithelial cells in
a band that corresponded to the germinative zone of the lens which is just anterior
to the lens equator. In another study, the
rabbit lens was wounded with a small
needle and one day later the 3H-thymidine
was injected into the anterior chamber.2
This study revealed that epithelial cells
around the wound edge incorporated 3Hthymidine. Since that time a number of
studies have been carried out on the details
of the 3H-thymidine incorporation into the
epithelial cells after lens wounding or
manipulation.3"5
Additional studies on the incorporation
of 3H-thymidine into lens epithelial cells
have been carried out on various species
and under various conditions. Hanna and
O'Brien,0 found that 3H-thymidine was
incorporated into the lens epithelial cells
located in the germinative zone of the
he use of tritium-labeled biochemical
intermediates together with the autoradiographic technique is a powerful tool for
the study of the physiological processes of
a cell. This technique is especially helpful
when only a few cells of a type are involved or where mixed cell populations
are to be studied. Studies on the lens with
the use of autoradiographic techniques have
centered around 3H-thymidine, an intermediate in deoxyribonucleic acid (DNA) synthesis. 3H-thymidine is incorporated into
the DNA of the lens epithelial cell during
the synthetic stages of cell division. Use
was made of this fact in two early reports
on 3H-thymidine uptake by lens epithelial
cells. Harding, Hughes, Bond, and Schork1
incubated the lenses from rats, frogs, and
young rabbits in Eagle's basal medium conFrom the Department of Pharmacology, University
of Arkansas Medical Center, Little Rock, Ark.
This investigation was supported by PHS Grants
NiB-04024 and NB-05076 from' the National
Institute of Neurological Diseases and Blindness,
United States Public Health Service.
480
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Volume 4
Number 4
Changes in DNA, RNA, and 'protein synthesis 481
adult rat lens. In addition, it was found
that these tritium-labeled epithelial cells
migrated posteriorly at a slow rate. In this
same study the rats fed a galactose diet for
12 days exhibited a seventeenfold increase
in the number of cells incorporating 3Hthymidine between the fourth and sixth
days. During this time the germinative
zone spread over the entire central area
of the lens and there was a corresponding
increase in number of cells incorporating
3
H-thymidine in the iris and ciliary processes. The influence of the age of the rat
on the lens epithelial cell incorporation of
3
H-thymidme was studied by Hanna and
O'Brien.7 It was found that the rate of
proliferation and migration of the epithelial cells was directly related to age. Brolin,
Diderholmh, and Hammar8 also utilized
3
H-thymidine to estimate the migration of
lens epithelial cells. The mouse and dog
lenses were found to have a germinative
zone very similar to that of the rat.9 In
the human the 3H-thymidine was found to
be incorporated into epithelial cells in a
similar spatial pattern in the lenses of a
teen-age child, a 35-year-old woman, and
in the extracted lenses from 7 elderly people with senile cataracts.
Cell division is composed of four major
stages each of which can be determined
in most cases with the use of 3H-thymidine.
For this determination the 3H-thymidine
is brought into contact with the dividing
cells and the cells are killed at various
times later. The cellular tritium uptake is
analyzed autoradiographically. By calculating the per cent of tritium-labeled cells at
one hour and the per cent of tritiumlabeled mitotic figures at intervals thereafter, it is possible to estimate the following: The period of DNA synthesis (S
phase), the premitotic interval (G2 phase),
the period of mitosis (M phase), and the
postmitotic gap (G:l phase). The cell cycle
has been estimated for a number of tissues
including the cornea and lens (reviewed
by Bertalanffy10). Thomson, Pirie, and
Overall11 attempted to do this with a small
group of 3- to 4-month-old rabbits. As
pointed out by the authors, the resulting
calculations are at best approximate because one animal was used at a critical
time period necessary in the calculations.
Mikulicich and Young12 used an adequate
number of 35-day-old rats and arrived at
an estimated cell cycle in the germinative
zone as follows: S phase lOVz hours, G2
phase minimum 2Vz hours, M phase between 2 and 5V2 hours, and Gx phase of
about 36 hours. Scullica, Grimes, and McElvain13'14 similarly studied several stages
of cell division in the 28- to 32-week-old
rat. They estimated the following cycle:
S phase 10 hours, G2 phase 5 hours, M
phase 1% hours, Gt phase 19 days (in the
germinative zone). These authors also
studied the effects of ionizing radiation on
the epithelial cells of the rat lens. They
found, as did Hanna and O'Brien15'1G an
initial inhibition followed by an overs wing
in the number of lens epithelial cells undergoing DNA synthesis. Also, these epithelial
cells moved a short distance before many
of the cells disintegrated with a loss of the
tritium-labeled DNA.
Experimental
Various species of animals were injected into
the anterior chamber with 5 nc of tritium-labeled
compound, 1 me. per milliliter, through a 33
gauge needle. Most of the animals were anesthetized with ether and topical lidocaine. The
animals were usually killed 2 hours later and the
enucleated eyes fixed in formalin or Carnoy's solution. The formalin-fixed tissues were paraffin embedded and cut into 5 /* thick sagittal plane sections. The capsule and epithelial cells of the
Carnoy's fixed lenses were flat mounted onto glass
slides. Both tissue preparations were treated with
xylene and hydrated through the alcohols, washed
in cold water, and painted with Kodak NTB3
liquid emulsion. After exposure for 10 to 60 days
in the dark the slides were developed in Kodak
D-19 developer, cleared with Kodak acid fixer, and
stained with hematoxylin and eosin.35 In studies
on the developing mouse lens the tritium-labeled
compounds were injected intraperitoneally. The
tritium-labeled compounds0 used are as follows:
adenosine (3 c. per millimole [c./mM.]), cytidine
°These compounds were purchased from the following
sources: New England Nuclear Corp., Boston, Mass.;
Nuclear-Chicago Corp., Des Plaines, 111.; Schwarz BioResearch, Inc., Orangeburg, N. J.; Volk Radiochemical
Co., Skokie, 111.
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Investigative Ophthalmology
August 1965
482 Hunna
(3 c./mM.), cytosine arabinoside (3 c./mM.),
deoxyadenosine (2.8 c./mM.), deoxycytidine (3
c./mM.), deoxyguanosine (2.8 c./mM.), deoxyuridine (1.3 c/raM.), Z-histidine (1.7 c./mM.),
hypoxanthine (1.7 c./mM.), 5-iodo-2'-deoxyuridine
(-6-3H, 0.34 c./mM.), Meucine (10 c./mM.),
orotic acid (-5-3H, 0.1 c./mM.), Z-phenylalanine
(1.6 c./mM.), Z-proline (3.2 c./mM.), thymidine
(0.8 to 8.8 c./mM.), and uridine (-5-3H, 1.7 to
13 c./mM., and -5,6-3H, 1.7 c./mM.).
Selected sections or flat mounts of lenses from
the various studies were treated with DNase or
RNase. 017
Unless otherwise indicated, the tritium-labeled
intermediates that gave only a nuclear label, or a
nuclear label at 45 minutes and a nuclear-cytoplasmic label at 2 hours represented DNA and
RNA labeling, respectively.
GERMINATIVE ZONE
NNULAR
PAD
Fig. 1. Schematic representation of chicken lens.
The dark arrows in the annular pad region refer
to the path taken by epithelial cells through the
pad and the number of weeks to arrive at each
point. The cross-hatched lines above the cell
nuclei indicate the cells undergoing RNA synthesis
as determined by 3H-uridine.
DNA metabolism
The germinative zone of the lens of
adult animals was located as a band of
epithelial cells undergoing both mitosis
and DNA synthesis. The germinative zone
in the reptilian type of lens was located in
a narrow band of epithelial cells just anterior to the annular pad (Fig. 1). This
type of eye was found to include the following: the Lacertilian eye, the chameleon
(Sauropsida); the Crocodilian eye, the 6•Worthington Biochemical Co., Freehold, N. J.
month-old American alligator (Caiman);
and the avian eye, the chicken (Gallus
domesticus, Leghorn).
A narrow germinative zone like that in
the human eye, i.e., located just anterior
to the lens equator and across from the
ciliary processes (Fig. 2) was found in the
following types of eyes: the Anuran eye,
the frog (Rana pipiens); the Placentalian
eye, the rabbit (Rodentia, Leporidae,
Oryctolagus); the rat and the mouse (Rodentia, Muridae, Rattus, and Muss); the
cat and the dog (Carnivora, Felis and
Canis) and the human. Two exceptions
were noted to this general classification of
the germinative zone. The lens of the
guinea pig (Caviodea, Caviidae) exhibited
a broad germinative zones while the minute lens from the American short-tailed
shrew (Blarina brevicauda) had no detectable germinative zone.
The cell cycle of the developing lens of
the Swiss mouse was determined with 3Hthymidine. Animals 3, 6, 12, and 24 days
old were injected intraperitoneally with 1
me. per kilogram of 3H-thymidine. The mice
were killed in the afternoons in the summer
time in groups of 6 at 1, 2, 5, 7, 12, and 17
hours later. Half of the eyes were fixed in
Carnoy's and the lens epithelial layer flat
mounted, and half of the eyes were fixed
in formalin and the lenses cut through the
sagittal plane. The center of the germinative zone was located in the flat mount
preparations and the per cent of cells
undergoing DNA synthesis and the per
cent of tritium-labeled mitotic figures
were determined at each time interval.
From a plot of these values versus time
the following phases of cell division were
calculated18: Gx phase, AQVz hours; S phase,
9 hours; G2 phase, 2V2 to 5 hours; M phase,
W2 to 4 hours; with a cell cycle of 56
hours (Fig. 2). No marked differences
were found in the cell cycle for the varying ages of the mice. The spatial distribution of the cells undergoing DNA synthesis
was determined on the sagittal plane sections and the results are given in Fig. 2.
In addition to the ages of the mice given
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Volume 4
Ntimber 4
Changes in DNA, RNA, and protein synthesis 483
Fig. 2. Graphic representation of the number and the average position of lens epithelial cells
undergoing DNA synthesis in mice of varying days (d) of age. The semicircle represents half
a sagittal plane section of a mouse lens with a nuclear bow region. The black dots represent
the average position of epitlielial cells undergoing DNA synthesis while the cross-hatched area
represents the range of cells found in DNA synthesis. The point at which epithelial cells become lens fibers is indicated by a dash and each 10 cells anterior to this are indicated by a
dash. The circle in the lower right-hand corner represents the length of a cell cycle (56 hours)
and the percentage of the total cycle taken up by the various phases of division.
in Fig. 2 those of the 2 day prenatal and
of the 1 and 180 day postnatal mice were
studied. DNA synthesis occurred in about
1 epithelial cell in 8 in the 2 day prenatal
lens. The germinative zone in the 1 day
postnatal mouse involved most of the epithelial cells except those in the premeridional row region near the lens equator.
This pattern of DNA synthesis continued
with a slight decrease in the per cent of S
phase epithelial cells until the eyelids began to open at 12 days of age. After this
time the germinative zone narrowed and
centered into a region just anterior to the
lens equator (180-day-old mouse). These
results on the spatial distribution of S
phase epithelial cells are similar to the
results reported by Hanna and O'Brien7
on the developing rat lens.
The mouse lens develops considerably
faster than does the human lens and the
lens development in the mouse is in the
embryonic stage until after birth. Therefore it is possible to study a number of
the developmental changes in the lens of
the mouse after birth. The developing
mouse lens from about 12 days after conception until the eyelids open at about 12
days of age corresponds to the period in
the human from the time when the lens
disc thickens until birth. In the mouse the
lens vesicle closes at 11 days and by 12%
days the lens vesicle detaches from the
surface epithelium and the lens capsule
begins to form. The posterior cells of the
lens vesicle grow and obliterate the lens
cavity by 13 days. From 13 days until birth
the nuclei of the primary fibers begin to
disappear to form the fetal nucleus which
is then covered by secondaiy lens fibers.
The newborn mouse lens, in development,
is equivalent to the 30 to 35 mm. long human embiyo after which the human fetal
period begins.19
The S phase and the M phase of cell
division were found to dissociate in the
12-day-old mouse lens. The S phase in this
lens (2 hour 3H-thymidine exposure) ex-
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Investigative Ophthalmology
August 1965
484 Hanna
tended from the meridional row cells to
the central area of the lens while the M
phase cells were in a narrow zone just
anterior to the meridional row cells. In
the zone of M phase cells, tritium-labeled
mitotic figures were found 5, 7, and 9 hours
after the 3H-thymidine injection. Only a
rare S phase cell in the central area of the
lens underwent mitosis by 17 hours after
the 3H-thymidine injection. These results
suggested that some of the epithelial cells
in the central area of the lens were arrested
in the G2 phase of cell division. Evidence
has been presented to show that the epithelial cells of the mouse are arrested in
the G2 phase of cell division." Results
obtained after lens wounding may indicate
a similar arrest of cells in the G2 phase.
Rats and rabbits were wounded with a thin
needle.2 3H-thymidine was injected into
the anterior chamber 18, 24, 30, and 36
hours after wounding and the animals
killed 2 hours later. At 18 hours the epithelial cells in the S phase were located
near the wound edge. By 24 hours the S
phase cells were located about halfway
between the wound edge and the lens
equator and M phase cells were located
just anterior to this. The M phase cells
were intermingled with the S phase cells
and the mitotic figure did not occur synchronously. This sequence of events is
clearly presented in the photomicrographs
of Harding, Feldherr, and Srinivasan.20 By
30 hours a second zone of S phase cells
appeared at the wound edge; however, by
36 hours no second zone of M phase cells
appeared. These results suggest that some
of the epithelial cells in the central area
of the lens had been arrested in the G2
phase of cell division and that after wounding more cells were arrested in the G2
phase. A number of authors have noted
the migration of lens epithelial cells by
utilizing 3H-thymidine and autoradiography. In each case a 2- to 4-month-old rat
or rabbit was studied for about 2 weeks.11"14
This migration of lens epithelial cells into
the lens cortex and the formation of lens
fibers have been studied in detail by Brolin,
Diderholmh, and Hammars and by Hanna
and O'Brien.0'7 In the very young rat the
epithelial cells migrate from the germinative zone and into the lens cortex to the
point where the lens fiber nucleus is lost
in about 8 weeks. By the time the rat is 5
months old this migration and differentiation into nonnucleated lens fibers take at
least 8 months and the migration of these
cells in the adult rat takes considerably
longer.7
A parallel exists between the rate at
which lens fibers develop and the rate at
which an ionizing radiation cataract develops.15' 10 This parallelism has not been
established for the reptilian eye since this
eye is very resistant to ionizing radiation.
In a cold-blooded reptile, the young (6
months) American alligator, a dose of
10,000 r of gamma rays from C0Co was used
without apparent effect on the transparency
of the lens over a 6 month period. A warmblooded animal with a reptilian type of
eye, the chicken, is also resistant to ionizing radiation. Pirie21'22 irradiated the chick
embryo (12 day) and adult chicken with
up to 6,000 r of x-rays and a cataract was
not observed in 6 months. It is possible
that the 12 day embryo is at a developmental stage that is resistant to cataract
development,23 but it is more difficult to
understand why the adult hen did not
develop cataracts. In the chicken lens many
epithelial cells are held in the annular pad
compared to the few epithelial cells in the
meridional rows in the rat lens, and it is
possible that x-ray-damaged chicken epithelial cells reach the lens cortex much
later. Six-week-old male Leghorn chickens
were injected into the anterior chamber
with 3H-thymidine and then killed 1, 2, 7,
14, 18, and 30 weeks later. Some of the
chickens at 14 weeks were given a second
injection of 3H-thymidine. In the lenses the
germinative zone cells migrated into the
annular pad at a slow rate as indicated in
Fig. 1. The second injection of 3H-thymidine into the 14-week-old chicken revealed
a 20 per cent reduction in the rate of lens
epithelial cell migration compared to that
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Number 4
Changes in DNA, RNA, and protein synthesis 485
of the 6-week-old chicken. It is roughly
estimated that the transient time of epithelial cells through the annular pad is on
the order of 18 months. Further, the chicken
lens was found to grow from the fifth
week on but the number of cells in the
annular pad decreased with time. Thus the
apparent rate of movement of epithelial
cells and the estimated time for the transit
of epithelial cells through the annular pad
can only be roughly estimated. In any
event, the time it takes for a chick epithelial cell to become a lens fiber is many
times longer than that for the young rat
or rabbit. Quite possibly the chicken lens
should be observed over a several-year period before it is concluded whether or not
radiation cataracts can be produced.
Several types of assault will induce the
lens epithelial cells to undergo a change
in DNA synthesis. Galactose-fed rats, for
example, were found to exhibit a marked
increase in the number of epithelial cells
undergoing DNA synthesis.0 Lens wounding will produce a similar effect.2 In both
of these examples the epithelial cells in the
meridional rows do not undergo DNA
synthesis. A study of the effects of cornea
freezing revealed an. interesting phenomenon. Rabbit corneas were frozen with
diy ice for 1 minute and 24 hours later
'H-thymidine was injected into the anterior
chamber. Sections of the eyes revealed
that lens epithelial cells in the meridional
rows and nucleated fibers in the nuclear
lens bow (superficial) underwent DNA
synthesis. This indicated that the developing lens fibers are capable of undergoing
DNA synthesis although these fibers did
not change shape during this process, nor
was the transparency of the lens altered
during the short observation period.
RNA metabolism
A study of the incorporation of 5-3H
uridine (15 me. per milligram intraperitoneally) was carried out in 1-, 3-, 6-, 12-,
24-, 96-, and 180-day-old Swiss mice. The
mice were killed 2 hours, 1, 3, 7, 14, and
12 days later in groups of three. This dose
of 3H-uridine intensely labeled the RNA
of heart, liver, lung, and kidney cells
while the RNA in cells of the eye was
minimally labeled. In none of the mice
studied was tritium-labeled RNA found in
lens cells 7, 14, or 21 days after the injection of 3H-uridine. Almost all nucleated
cells of the lens contained tritium-labeled
RNA in the 1- and 3-day-old mice 2 hours
after the injection of 3H-uridine. In these
lens cells the tritium was localized in or
around the cell nucleus. By 1 and 3 days
after the 3H-uridine injections the tritiumlabeled RNA was found throughout the
cell and the grain count per cell was much
reduced at 3 days. In mice older than 3
days of age, the tritium-labeled RNA was
found less often in the more mature lens
fibers of the cortex as the age of the animal increased. The tritium-labeled RNA
was found only in the epithelial cells and
in the most superficial lens fibers of mice
older than 24 days of age (Fig. 3). In the
adult mouse, rat, rabbit, and senile cataractous human lenses after 2 hours' exposure to 3H-uridine, the tritium-labeled RNA
was found in all epithelial cells and in only
the newly forming fibers in the lens equator
region (Fig. 3).
The pattern of RNA synthesis in the
lens epithelial cells was studied in lens
flat mount preparations of the rat and
human. The lenses were incubated in
Eagle's basal medium containing 3H-uridine for 2 hours. It was found that the
epithelial cells over the central area of the
lens were undergoing a uniform amount of
RNA synthesis (Fig. 4). The cells in the
germinative zone were synthesizing a similar amount of RNA, but the cells in the
meridional rows were undergoing a reduced amount of RNA synthesis. The newly
forming fibers at the edge of the lens epithelium, however, were undergoing a high
rate of RNA synthesis. This fact is graphically illustrated in Fig. 4 where 3H-leucine
was utilized but the results were similar to
those obtained with 3H-uridine.
One day after lens wounding there were
a large number of epithelial cells in the
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486 Banna
EPITHELIUM
Fig. 3. Graphic representation of RNA metabolism
in the lens of an adult rat eye. Two hours after the
injection, into the anterior chamber of 5-3Huridine, the tritium label was found in and around
the nuclei of cells covered by the cross-hatching.
By 1 day the tritium was found throughout these
same cells with the highest concentration in the
newly developing lens fibers as indicated by the
dark cross-hatching in the equator region.
Investigative Ophthalmology
August 1965
M phase of cell division. Flat mount preparations of these lenses were found useful
in the study of RNA synthesis in relation
to the mitotic stages. 3H-uridine was injected into the anterior chamber of rat eye
given a lens wound 1 day before. Lenses
were removed 45 minutes and 2 hours
later. An examination of the flat mount lens
preparation revealed that about half of the
anaphase and metaphase cells were undergoing RNA synthesis at 45 minutes, whereas
all mitotic phases contained tritium-labeled
RNA at 2 hours. Therefore, RNA synthesis
in the lens epithelial cell is a continuous
process except for a short interval during
anaphase and metaphase.
A comparison was made of the incorporation of 3H-uridine into the RNA of the
various cells of the eye. A dose of 30 me.
per kilogram of 3H-uridine (13 c./mM.)
was injected intraperitoneally into 24-dayold mice. The mice were killed 2 hours
Fig. 4. Autoradiograph of a flat mount preparation of epithelial cells from a senile cataract
human lens after 2 hours' incubation in Eagle's basal medium containing 5-3H-uridi.ne. The
black dots and area over and around each cell nucleus are an indication of the extent of RNA
synthesis in these cells. Note the nearly uniform amount of RNA synthesis in this central area
of the lens epithelium. (x400; reduced %.)
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Volume 4
Number 4
Changes in DNA, RNA, and protein synthesis 4S7
later and the eyes prepared for autoradiographic analysis. The exposed silver grains
in the photographic emulsion over individual cells were counted and averaged. The
results of this study are given in Table I. It
can be seen from the table that the developing lens fibers incorporated the greatest
amount of 3H-uridine into RNA of all cells
in the globe of the eye. Autoradiograms of
cells from the liver, heart, spleen, and kidneys taken from these same animals contained so much tritium-labeled RNA that
the emulsion was overexposed after a time
of exposure equivalent to that used for the
eye tissues.
In the adult mouse, rat, rabbit, and human lens the penetration of 3H-uridine into
the older lens cortex fibers was too slow to
obtain information on the RNA synthesis
of lens fibers nuclear extrusion. The young
chicken (8 weeks) was found to be very
useful for this study because the 3H-uridine
Table I. Grain count over individual cells
of various parts of the mouse eye (24 days
old) 2 hours after the intraperitoneal injection of 3H-uridine or 3H-leucine (average
of 4 eyes each)
UridineRNA
Leucineprotein
Lens:
Epithelium, central
equator
Cortex, equator
anterior
posterior
Nucleus
7
8
22
0
0
0
24
30
40
0
0
0
Cornea:
Epithelium
Stroma
Endothelium
12
2
2
26
20
14
Iris
10
24
Ciliary processes
11
24
Eyelid
11
21
Coroid
9
28
Ocular muscle
16
22
Retina:
Ganglion cells
Inner nuclear layer cells
12
32
21
Area of eye
5
readily penetrated into these lenses. To
establish the diffusion of 3H-uridine into
the chicken lens, the tritiated compound
was injected into the anterior chamber
with the least possible mixing. The lens
taken at 2 hours showed a much greater
intensity of tritium-labeled RNA in the
anterior half of the lens than in the posterior half. This information was of importance because the nucleus of lens fibers
in the process of nuclear extrusion was
located in the anterior half of the lens.
These cortex fibers in the process of differentiating into nuclear fibers were found in
several progressive stages of nuclear disintegration. An early stage was located
with a loss of nuclear granular stain. At a
later stage about one-half of the nucleus
did not stain and further toward the lens
nucleus the fibers were found with a small,
darkly staining mass in place of a nucleus.
None of these cells were found to be undergoing a measurable amount of RNA
synthesis.
Protein metabolism
Several amino acids with high specific
activity and stability of tritium label are
available for studies on protein synthesis.
Of these 4,5-3H-Z-leucine was especially
useful because this amino acid is generally
incorporated into proteins.24 Four amino
acids were chosen for this study. Leucine
and 3H-ring-Z-phenylalanine were chosen
as representative of the more hydrotropic
amino acids. Levohistidine (3H) and 5-3HJ-proline were chosen as representative of
the more lipotropic amino acids. However,
histidine and praline are to some extent
converted into other compounds besides
being incorporated into protein. The amino
acids were injected into the anterior
chamber of rats and the animals were
killed 2 hours later. The tritium label when
found was present in all parts of the nucleated cell. Leucine ( 3 H) and 3H-phenylalanine gave a similar pattern of lens protein incorporation in the epithelial and
cortex fiber cells (Figs. 5 and 6 and Table
I). The distribution of tritium from these
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Inoestigatioe Ophthalmology
August 1965
488 Hanna
Fig. 5. Autoradiograph of lens epithelial cell from a flat mount preparation of rat lens removed
2 hours after the anterior chamber injection of 3H-leucine. The slide was destained and therefore the intensity and frequency of the black dots give an indication of the rate of protein
synthesis in these cells. The right-hand side of the figure indicates a high rate of protein
synthesis in a narrow band of epithelial cells difFerentiating into lens fibers. (x300; reduced V&.)
EPITHELIUM,
GERMINATIVE
ZONE
CAPSULE
Fig. 6. Graphic representation of protein synthesis
in adult rat lenses. Cross-hatched area represents
protein synthesis 2 hours after the injection into
the anterior chamber of 3H-leucine. When 3 Hhistidine and 8H-proline were used, the indication
of protein incorporation of these two amino acids
is indicated by the heavy cross-hatched lines. The
lens capsule above the epithelial cells was found
to contain a small amount of tritium also.
two amino acids was very similar to that
obtained with 3H-uridine. The developing
mouse lens was studied in a manner similar
to that for 3H-uridine except SH-Ieucine
(10 me. per kilogram) was used. The results obtained with 3H-leucine were very
similar to those with 3H-uridine except
proteins were labeled in nucleated cells
of the lens of mice. In general, protein synthesis occurred in those cells that were also
undergoing RNA synthesis.
Histidine ( 3 H) and 3H-proline gave results similar to those of Ieucine on the rat
lens except these 2 amino acids were incorporated into the protein of the newly
developing lens fibers to a much greater
extent (Fig. 7). Also, aH-histidine was incorporated into protein over the posterior
part of the lens at 2 hours while 3H-proline
was intensely incorporated into the ganglion cells of the retina.
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Volume 4
Number 4
Changes in DNA, RNA, and protein synthesis 4S9
Fig. 7. Autoracliogram of sagittal plane
the anterior chamber of 3H-histidine.
indicated by the blackness of the area.
of the autoradiograph since the area
reduced %.)
lens section of rat killed 2 hours after an injection into
The rate of protein incorporation of 3H-histidine is
A torn lens section was chosen to show the resolution
without tissue underneath it is almost clear. (x250;
Applications
The potential of lens epithelial cells to
utilize various metabolic intermediates and
related compounds was studied in the rat
and human lens. Adult rats were injected
in the anterior chamber with various tritium-labeled compounds and the animals
were killed 45 minutes and 2 hours later.
The lenses were sectioned or made" into
flat mount preparations for autoradiographic analysis. With the compounds studied four general types of autoradiographic
grain patterns were obtained: (1) The
grains were located directly over the cell
nucleus of epithelial cells in the germinative zone. This DNA pattern was produced
by tritium-labeled thymidine, IDU, deoxyuridine, deoxycytidine, deoxyadenosine.
(2) The grains were directly over the
nucleus at 45 minutes and over the entire
cell at 2 hours. This RNA pattern was
found over most nucleated cells of the lens
and it was obtained with tritium-labeled
5-8H-uridine, and 5-8H-orotic acid. (3) The
grains were over the entire cell at 45 minutes and at 2 hours. The protein type of
labeling pattern was produced by tritiumlabeled leucine, phenylalanine, histidine,
and proline. (4) A mixed RNA and DNA
grain pattern was found with tritiumlabeled cytidine, adenosine, guanosine, 5,63
H-uridine, 8-3H-hypoxanthine and deoxyguanosine. The deoxyguanosine in aqueous
solution was stored in a frozen state and
it is possible that some decomposition occurred in preparing the material for injection. A similar pattern of tritium-labeled
cells occurred with these compounds in
the cells of the cornea. A similar study was
carried out on the human senile cataract
lens except the lens was incubated in
Eagle's basal medium containing the tritium-labeled compound before the lenses
were sectioned or flat mounted. Results
obtained with the human lenses were like
those obtained with the rat lenses. These
studies on the rat lenses and the human
senile cataract lenses indicated that the lens
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Investigative Ophthalmology
August 1965
490 Hanna
epithelial cells underwent a similar metabolic process.
Several "antimetabolites" of nucleic acid
intermediates were studied in the rat lenses
after anterior chamber injections. Half of
the lenses were flat mounted and half were
sectioned. The following results were obtained: 3H-IDU was incorporated into
DNA, this labeled DNA appeared in the
mitotic figures, and these cells migrated
to the nuclear bow region. Cytosine 3Harabinoside gave a mixed DNA and RNA
label like that obtained with 3H-cytidine
while 5-3H-fluorouracil gave only an RNA
label. Tritium-labeled 6-azauridine gave
no labeled cells in the eye. Iodine-131 has
been noted by Wheeler, Harding, and
Hughes25 to be incorporated into the germinative zone of cells after lens wounding
after in vitro incubation with 131I-IDU
medium. In repeating this experiment with
3
H-IDU in place of the 131I-IDU, the same
results were obtained. This established that
the IDU was directly incorporated into
DNA.
Summary
Cell division in the developing mouse
lens was studied by thymidine-tritium and
autoradiographic techniques. From the
time the secondary lens fibers began to
form just before birth until the eyes opened
at about 12 days, most of the epithelial
cells were undergoing almost continuous
cell division. The cell division cycle was
estimated to be 56 hours during this period.
Mouse eye development at the time the
lids open corresponded in development to
that of a human eye at birth. The number
of epithelial cells dividing over the lens
decreased markedly after the eyes opened.
Evidence was presented that indicated
some of the cells in the central area of the
lens rested in the G2 phase of cell division.
Cell migration in the young rat lens
from the germinative zone to the point
where nonnucleated lens fibers formed took
about two to four months. It would take an
estimated one-half to one year for lens
epithelial cells to migrate this distance in
the adult rat lens.
Cell division in the chicken lens was
found in a narrow zone just anterior to the
annular pad. These epithelial cells migrated through the annular pad at a slow
rate with an estimated transient time of
one and one-half years.
Ribonucleic acid (RNA) and protein
metabolism in the developing mouse lens
was studied with the use of uridine-tritium
and leucine-tritium, respectively. Nucleated
cells undergoing RNA and protein metabolism were found throughout the lens of the
newborn mouse and this continued until
about 6 days of age. Lens fiber cells in the
process of losing their cell nuclei were not
undergoing RNA or protein synthesis except in the very young animals through
6 days of age. This was most easily observed in the young chicken lens where the
cortex fibers differentiating into nuclear
fibers involved a number of cells which
do not undergo RNA and protein synthesis.
With increasing age of the mouse the
uridine-tritium or leucine-tritium did not
readily penetrate deep into the lens cortex.
In the adult lens of various species, including the senile cataract lens of the human, uridine-tritium and leucine-tritium
were found in epithelial cells and in developing fiber cells at the equator region.
Tritium from leucine-tritium but not from
uridine-tritium was found in the lens capsule.
The help of the following people in various
aspects of this project is gratefully acknowledged:
Travis W. Jenkins, Henry C. Keatts, John E. Slayden, and Katherine P. Wilkinson of the Department of Pharmacology, University of Arkansas
Medical Center.
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B. D.: Incorporation of thymidine by injured
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Volume 4
Number 4
Changes in DNA, RNA, and protein synthesis 491
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