Investigative Ophthalmology & Visual Science, Vol. 30, No. 3, March 1989
Copyright © Association for Research in Vision and Ophthalmology
Reports
Aging and Rotes of Lens-Cell Differentiation In Vivo,
Measured by o Chemical Approach
Richard J. Cenedello
cells which become labeled following brief exposure
to 3H-thymidine can be followed by measuring the
distribution of 3H-labeled DNA between the lens
capsule (epithelial cell layer) and lens body (fiber
cells) with time after injection. Since terminal differentiation is required for the labeled cells to move
from the capsule to the lens body, measurement of
the rate of this movement can provide an estimate of
the rate of epithelial cell differentiation.
The current study uses a modification of the previously described chemical approach for quantitating
rates of epithelial cell differentiation in vivo.10 A suspicion that not all TCA-insoluble radiolabel directly
recovered from the lens following injection of rats
with 3H-thymidine was DNA, led us to compare the
recovery of labeled DNA by direct TCA precipitation
with that recovered following protein digestion and
phenol extraction of the DNA. Indeed, not all TCAinsoluble radiolabel directly recovered from the lens
was DNA, and thus the revised approach permitted a
more accurate assessment of rates of differentiation
and DNA synthesis.
Materials and Methods. Animal use was approved
by the institutional animal care committee and all
experimental procedures reported here conformed to
the ARVO Resolution on the Use of Animals in Research. Sprague-Dawley rats (Hilltop Lab Animals,
Scottdale, PA) of 6 to 40 days of age were injected
(i.p.) with 4 MCi/g b.w. of 3H-(methyl) thymidine (2
Ci/mmol, New England Nuclear, Boston, MA). Rats
were sacrificed at various times in groups of four to
eight over the subsequent 2 to 3 weeks and the capsule (epithelial cell layer) and lens body (fiber cells)
were separated as described before.10 Pairs of capsules
and lens bodies from individual rats were homogenized for 2 min with a tissumizer (Tekmar Co., Cincinnati, OH) at 4°C in 1.0 ml of 0.9% NaCl containing 0.4 mg of DNA carrier (salmon, type III, Sigma,
St. Louis, MO). The homogenates were combined
with two separate 1 ml saline washes of the homogenizer probe, protein was digested at 37°C for 3 hr by
We describe a direct and comparatively rapid chemical approach for quantitating rates of lens epithelial cell differentiation in vivo and apply it to a study of the basis of the
precipitous decrease in the rate of lens growth in the rat
between about 1 and 6 weeks of age. Rates of terminal
differentiation of epithelial cells into fiber cells were quantitatively described by a first-order-fractional rate constant
(k) for loss of 3H-DNA (labeled from injected 3H-thymidine) from the capsule (epithelium) to fiber cells. The rate
constant (expressed in days) was calculated using the halflife estimated from semilogarithmic plots of the percent of
total lens 3H-DNA measured in the capsule fraction versus
time after injection of 3H-thymidine. The rate constant decreased from about k = 0.18 day"1 at 6 days of age to about
0.09 day"1 at 18-20 days of age and changed little thereafter. The decrease in lens growth at very early ages in the rat
is at least partially due to decreases in rates of epithelial cell
differentiation. Invest Ophthalmol Vis Sci 30:575-579,
1989
Although the lens grows throughout life,1 the rate
of growth is much higher during early development
and it decreases rapidly with aging to a steady state.2
The decrease in the rate of lens growth with aging has
been attributed to a reduction in the proliferation of
lens epithelial cells.3 The decrease could also be partially due to changes in rates of epithelial cell differentiation. Differentiation in the lens has been studied
by autoradiographic technics which involve microscopically following the migration of labeled epithelial cells from the lens capsule to the lens body after
injecting animals with 3H-thymidine.2"7 The autoradiographic approach is limited by being essentially
semiquantitative and highly time-consuming. The
present work uses a direct chemical approach to reexamine aging-related changes in rates of growth of the
rat lens that is based upon following the movement of
radiolabeled DNA from epithelial to fiber cells.
DNA synthesis in the ocular lens is confined to the
epithelial cells.8-9 Substrate is available to these cells
for DNA synthesis for only a few hours after intraperitoneal (i.p.) injection of rats with 3H-thymidine.10
Thus, the fate of a discrete population of epithelial
575
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1989
100 Mg of added proteinase K (Boehringer Mannheim, Indianapolis, IN) and DNA was extracted with
an equal volume of phenol (equilibrated with 1 M
tris, pH 8): chloroform (1:1). The aqueous phase was
recovered after centrifugation and extracted twice
with ether; the residual ether was blown off under
nitrogen and the DNA precipitated by adding an
equal volume of 20% TCA. The TCA precipitate was
recovered, washed and prepared for counting as described before.10 No protein could be detected in the
lens fractions following treatment with proteinase K.
The affect of age upon the availability of substrate
for DNA synthesis was examined by sacrificing 6, 11,
20 and 40-day-old rats at 1 hr after injecting (i.p.)
3
H-thymidine (4 fiCi/g b.w.). Lenses were homogenized in 3 ml saline containing 0.4 mg of DNA carrier, 0.6 ml of 60% TCA was added and the samples
were held overnight at 4°C. Samples were then centrifuged and the supernatant recovered. Aliquots of
the supernatant were assayed for radioactivity before
and after evaporation to dryness. We previously
found that 3H-thymidine largely accounted for the
radioactivity remaining after evaporating the TCAsoluble fraction from lenses of rats injected 1 hr earlier with 3H-thymidine.'° Tritiated water accounted
for the volatile component in the total TCA-soluble
radioactivity.
Results. In our initial study,10 3H-labeled DNA was
recovered by directly homogenizing the lens fractions
in TCA followed by TCA and ethanol washing of the
precipitates. We, like others," assumed that the
TCA-insoluble 3H thus recovered after exposure to
3
H-thymidine was 3H-labeled DNA. Our previous
observation10 that the ratio of TCA-insoluble 3H in
the epithelial cell layer to lens body was often only
about two to three to one at 24 hr after injection of
3
H-thymidine into the rat, led us to suspect that some
of the TCA-insoluble radiolabel directly precipitated
from the lens body was not 3H-labeled DNA, because
labeled epithelial cells should not have left the capsule
by differentiation at this early time. We now recognize that this direct precipitation of the homogenates
results in recovery of some TCA-insoluble 3H from
the fiber cell mass (lens body) that is not 3H-DNA
(Table 1). Small amounts of 3H-thymidine, a labeled
metabolite or an impurity appear to become tightly
associated with the concentrated proteins of the lens
fiber cells, since treatment of the lens homogenates
with proteinase K removed much of the TCA-insoluble 3H obtained by direct precipitation (Table 1).
Note that treatment of the homogenized capsule
fraction, which contains comparatively little protein,
with proteinase K had no effect on the recovery of the
TCA-insoluble 3 H.
Vol. 30
Rates of differentiation were estimated from firstorder decay curves generated by semilogarithmic
plots of the percent of total lens 3H-DNA measured
in the capsule against time after injection of 3H-thymidine (Fig. 1A). This expression accurately describes a first-order process. The fractional rate constant was calculated from the equation T, /2 = 0.693/
k, where the half-life for loss of 3H-labeled DNA from
the capsule was directly estimated from the experimentally generated decay curves. This rate constant
is expressed in days and can represent the percent of
the labeled epithelial cells differentiating per day at a
given time. In our earlier work,10 we estimated rates
of epithelial cell differentiation from decay curves
generated by semilogarithmic plots of the ratio of
TCA-insoluble 3H in the capsule to lens body against
time after injection. We now recognize that changes
in this ratio can poorly describe a given first-order
process and can result in overestimation of the magnitude of the rate constant.
At all ages studied, 90% or more of the total ^ - l a beled DNA in the lens was recovered from the capsule fraction prior to the onset of differentiation of
labeled epithelial cells (Fig. 1A). About 3 days were
required in the 6- and 12-day-old rats for labeled epithelial cells (3H-DNA) to migrate from the proliferative zone of the capsule to the meridional zone where
they began to differentiate (leave the capsule) (Fig.
1A). This migration required about 5 days in the
larger lenses of the 18-20-day-old rats and about 9
days in the 40-day-old rats. The longer migration
times in the older animals presumably reflect the
greater distances which the labeled cells must traverse
in the larger lenses to reach the zone of differentiation. The first-order rate constant calculated from the
decay of 3H-DNA from the capsule provides an estimate of the rate of terminal differentiation of the lens
epithelial cells. The differentiation rate constant (k)
estimated for the 6-day-old rats was about 0.175
day"1; that is, once the labeled cells began to differentiate, about 17.5% of this cell population were differentiating per day (Fig. 1 A, B). The value decreased to
about 0.13 day"1 by 12 days of age, then to about
0.085 day"1 by 18-20 days of age and thereafter it
changed little (Fig. 1A, B). The rate constant estimated for the 6-day-old rats could be low since some
continued synthesis of labeled DNA appears to have
occurred over the 2-week interval following injection
of 3H-thymidine into 6-day-old rats (Fig. 2A). The
level of 3H-DNA increased by about 25% over this
interval.
Incorporation of 3H-thymidine into DNA of the
whole lens decreased with age (Fig. IB). Incorporation at 40 days of age was about 40% of that at 20 days
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No. 3
Table 1. Recovery of 3H-labeled "DNA" from fractions of rat lens by direct TCA precipitation versus recovery
after treatment with proteinase K and phenol extraction
Treatment
Days after
injecting
3
H-thymidine
Age (days) at
injection with
3
H-thymidine
Lens
fraction
Proteinase K and
phenol extracted
Direct TCA Ppt
6
6
2
2
1340
711
1292
174
Capsule
Lens body
6
6
10
10
308
2811
308
1516
Capsule
Lens body
40
40
to to
DPM/2 capsules or 2 lenses
Capsule
Lens body
551
774
572
58
Capsule
Lens body
40
40
15
15
208
2173
209
278
Pools of four to six capsules (lens epithelium) or lens bodies (lens minus
the capsule) were separately homogenized in saline containing 0.4 mg of
carrier DNA. Half of each homogenate was directly precipitated with TCA
(Direct TCA Ppt) and the other half was treated with proteinase K, extracted
with phenolxhloroform (1:1) and then precipitated with TCA (Phenol extracted). All TCA precipitates were washed three times with 10% TCA and
twice with ethanol prior to measuring their tritium content. Each value is the
mean of duplicate pools.
of age and only about 20% of that at 6 days of age.
The decreased incorporation of 3H-thymidine into
lens DNA generally paralleled the decreased rate of
lens growth over this age interval (Fig. 1C). However,
part of this apparent decrease might be related to
lower levels of substrate (3H-thymidine) present in
<'
z6
k>.
40
C
Z
20
6 Days Old
o
0
I
2
3 4
5 6
7
8
9
\
10 II
12 13 14 15 16 17 18 19 20 21 22
Days After Injection of
3
H Thymidine
14
22
30
Age (days)
3
Fig. 1. (A) First-order decay curves for loss of H-DNA from the lens capsule (epithelial cell layer) to the lens body (fiber cells) in rats
injected at different ages with 3 H-thymidine. Each point is the mean ± SEM for four to eight rats. Curves were fit by computer; all correlation
coefficients (r) were 0.98 or greater. (B) Changes in the fractional rate constant and incorporation of 3 H-thymidine into DNA versus age.
Fractional rate constants (k) were calculated from the half-lives estimated from the decay curves presented in (A) (k = 0.693/T, /2 in days).
3
H-DNA levels are the mean incorporation ± SEM of all the rats within each age group (28 to 55 rats per group) over the time periods studied.
(C) Changes in lens wet weight with aging. Each value is the mean ± SEM of a minimum of six pools of lenses with a minimum of 14 lenses per
pool.
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578
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1989
D
900
400
300
1200
1100
1000
900
800
-c
si
-
T
1
T
T
/
rf4y
1300
1200
1100
—
1000
900
2000
1900
~A
1800
1700
1600
ISOO
i
1400
i
i
i
i
i
i
i
II
13
IS
17
19
21
23
DAYS POST INJECTION
Fig. 2. Changes in total lens content of 3 H-DNA. Dashed lines
indicate the average dpm of 3 H-DNA per pair of whole lenses over
the time periods studied following injection of 3 H-thymidine into 6
(A), 12 (B), 18-20 (C) and 40 (D) day-old rats. Points and bars are
the mean ± SEM of individual pairs of lenses (n = 4 to 8) at each
time. Solid lines are slopes fitted by computer. One way analysis of
variance was used to test whether the slopes were significantly
different from zero. The slopes in (A) and (B) were significantly
different from zero at the 0.003 and 0.048 levels, respectively.
Slopes in (C) and (D) were not significantly different from zero.
the lenses of the older animals following i.p. injection
of 3H-thymidine. The concentration of nonvolatile
and TCA-soluble radioactivity, 3 H-thymidine,'°
present in the lens at 1 hr after injection was similar
for 6- and 11-day-old rats but was markedly less in
lenses of 20- and 40-day-old rats relative to those of
the younger animals (Table 2). A smaller but still
significant difference was seen between the level of
labeled substrate in the aqueous humor of 11- versus
40-day-old rats at 1 hr after injection; 1.43 ± 0.10
Table 2. Effect of age upon availability of substrate
for lens DNA synthesis
DPM X 10 6/g lens (wet wt)
Rat age *
(days)
n
Total
TCA-soluble
6
11
20
40
6
6
5
5
3.975 ±0.197
4.018 ±0.197
3.663 ±0.168
4.195 ±0.138
TCA-soluble
and
nonvolatile
1.329
1.168
0.536
0.275
±0.030
±0.066
±0.019
± 0.020
* Rats were injected (i.p.) with 4 <jCi/g b.w. 3H-thymidine (2 Ci/mmol)
and sacrificed 1 hr later.
Values are mean ± SEM. TCA-soluble and nonvolatile radioactivity is
largely 'H-thymidine.10 Tritiated water accounts for the difference between
total TCA-soluble and TCA-soluble, nonvolatile.10
Vol. 30
X 106 dpm of nonvolatile and TCA-soluble 3 H/ml at
11 days of age versus 0.65 ± 0.12 X 106 dpm/ml at 40
days.
Discussion. Rates of differentiation of lens epithelial cells in the rat were highest at 1 week after birth
and then decreased to a steady state rate at about 3
weeks of age. The very rapid growth of the rat lens at
6 days of age could be due to both the rapid differentiation of these cells (after they reach the meridional
zone the epithelial cells of the very young rat are
converted to fiber cells much more rapidly than at
older ages) and to rapid proliferation of epithelial
cells as reflected by the apparent high rate of DNA
synthesis. The decrease in the rate of lens growth between 1 and 3 weeks of age could be due to marked
decreases in both rates of cell differentiation and proliferation.
In older animals, cell proliferation might be the
prime determinant of lens growth, since DNA synthesis appeared to decrease sharply between about 3
and 6 weeks of age while the rate of differentiation
remained essentially constant during this period (Fig.
IB). However, one must be cautious in equating
changes in incorporation of tritium into lens DNA
following injection of 3H-thymidine into intact animals to changes in true levels of DNA synthesis and
thus epithelial cell proliferation. Since incorporation
of 3H-thymidine into lens DNA could depend upon
the concentration of 3H-thymidine reaching the lens,
our finding of lower levels of labeled substrate in the
lenses of older rats at 1 hr after injection (Table 2)
suggest that a decrease in substrate availability might
account for some of the reduced incorporation of
3
H-thymidine into lens DNA with aging (Fig. IB).
Perhaps the rate of metabolism of injected 3H-thymidine is directly related to body size. In view of this
possibility, any conclusion from our data on the relative importance of changes in epithelial cell differentiation versus proliferation in explaining net changes
in rates of lens growth with aging must be tentative.
The differentiation rate constant of 0.13 day"1 determined for the 12-day-old rat is much lower than
the 0.27 day"1 value we reported earlier.10 The earlier
value was estimated from changes in the ratio of
TCA-insoluble 3H in the capsule to lens body with
time after injection of 3H-thymidine. We now recognize that this relationship can poorly describe a firstorder process and also that much of the total 3 H label
directly precipitated by TCA from the fiber cell mass
in the earlier study might not have been 3H-labeled
DNA (Table 1).
Using autoradiographic technics, Mikulicich and
Young concluded that epithelial cells of the lens proliferative zone are replaced by migration and differentiation every 3 to 4 days in 6-day-old rats.5 Based
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Reports
upon our current finding that the differentiation rate
constant is equal to about 0.18 day"1 in 6-day-old
rats, we estimate that about 8 days would be required
to replace 75% of the proliferative epithelial cells at
this age. This rate could be low because of continued
entry of 3H-DNA into the epithelial cell compartment (capsule) due to delayed synthesis (Fig. 2A).
Delayed DNA synthesis could result in a 25 to 30%
underestimation of the rate constant at 6 days of age.
Thus, the true rate of differentiation at this age could
be close to about 0.25 day"1, a value in better agreement with the observation of Mikulicich and Young.5
One should realize, however, that delayed synthesis
of 3 H-DNA following injection of 3 H-thymidine
could also complicate the measurement of epithelial
cell displacement and differentiation by the autoradiographic approach. Continued synthesis of 3 HDNA in the lens after pulse injection of 3H-thymidine
could result from the delayed availability of labeled
substrate derived from degraded DNA of rapidturnover cells, such as those of the gastrointestinal
tract.
Key words: lens, differentiation, aging, DNA synthesis, epithelial cell
Acknowledgments. The author thanks Mrs. Nancy Waletzko for her excellent technical assistance, Dr. Bharat
Pandya for his advice on DNA recovery, Dr. Donald
Kangas for assistance with the statistical analyses and Mr.
David Cenedella for assistance with computations.
From the Department of Biochemistry, Kirksville College of Osteopathic Medicine, Kirksville, Missouri. Supported by NIH grant
EY-20568, Bethesda, Maryland. Submitted for publication: March
579
11, 1988; accepted October 5, 1988. Reprint requests: Richard J.
Cenedella, Department of Biochemistry, Kirksville College, of Osteopathic Medicine, Kirksville, MO 63501.
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