the effect of light deprivation on the mouse lens
TRANSCRIPT
Exp. Eye Res. (1998) 66, 669–674
The Effect of Light Deprivation on the Mouse Lens
ROBERT C. AUGUSTEYN
National Vision Research Institute of Australia, 386 Cardigan Street, Carlton, Victoria 3053, Australia
(Received Oxford 18 December 1997 and accepted in revised form 27 January 1998)
This work was undertaken to test the hypothesis that first exposure of the eye to light is responsible forthe changes in lens protein expression patterns observed around the time of birth.
The effect of light deprivation on lens properties was examined in Balb c mice which were bred, rearedand maintained in complete darkness for up to 7 months. Data were collected on body weight, lensweight, lens protein contents and crystallin distributions. The data were compared with those obtainedfrom age matched mice maintained in natural light}dark conditions.
No significant differences were observed in body weight between animals maintained in the light anddark. However, animals kept in the dark had significantly smaller lenses. After 6 months in the dark, thelens represented 0±02% of body weight compared with 0±031% in the light reared animals (P!0±001).Lens protein concentration, insoluble protein contents and crystallin synthesis patterns wereindistinguishable for the two groups of animals.
It is concluded that light stimulation of the eye is required for optimal lens growth but does not affectthe production of specific crystallins. # 1998 Academic Press Limited
Key words : light deprivation; lens weight ; crystallin distribution; mice; growth factors.
1. Introduction
There is considerable interest in the effect of light and
visual experience on the growth and functioning of
the eye. Much of this interest is concentrated on the
role of visual stimuli in the development of the eye’s
refractive properties. Visual deprivation can produce
severe myopia in chicks, humans, monkeys and tree
shrews (Mutti, Zadnik and Adams, 1996). This
appears to be due, mainly, to ocular elongation rather
than to changes in the major refracting structures, the
lens and cornea. However, there may also be sec-
ondary effects of visual deprivation on these tissues.
These have not been addressed.
In previous studies on the ontogeny of human and
bovine lens proteins, we had observed that there are
substantial changes in the patterns of proteins synthe-
sized by the lens at around the time of birth
(Pierscionek and Augusteyn, 1988; Thomson and
Augusteyn, 1985). In particular, we had noted that
the synthesis of γ-crystallins ceases at around this time
while that of the closely related γs(β
s)-crystallin
commences, albeit at much lower levels. As a result,
proteins in the cortex of the postnatal lens are
comprised mainly ("90%) of the polymeric α- and β-
crystallins while the nuclear tissue which is of prenatal
origin, contains substantial amounts (up to 30%) of
the monomeric γ-crystallins. The significance of these
changes in terms of lens structure and function is still
not clear at the present time. It may be that the low
Correspondence should be addressed to: R. C. Augusteyn,National Vision Research Institute of Australia, 386 CardiganStreet, Carlton, Victoria 3053, Australia.
molecular weight γ-crystallins are involved in the
processes which result in the loss of water and
consequent tissue hardening observed in the nucleus
of the adult lens (Pierscionek and Augusteyn, 1988;
1992; Slingsby, 1985). Their absence from the lens
cortex would ensure that this tissue remains soft and
pliable.
The triggers for the changes in protein synthesis
patterns have not been identified. On the basis of our
previous observations (Pierscionek and Augusteyn,
1988; Thomson and Augusteyn, 1985) we proposed
that they may involve growth factors released by the
retina in response to the sensory stimulation resulting
from first exposure to light, but little was known about
possible factors at the time. Since then it has been
discovered that cytokines such as the Fibroblast
Growth Factors (mainly bFGF), insulin and Trans-
forming Growth Factors (mainly TGFβ2) are able to
influence the proliferation, migration and differen-
tiation of lens epithelial cells on capsular explants in
culture (de longh and McAvoy, 1992; Schulz, Cham-
berlain and McAvoy, 1996). All are present in the eye
where bFGF and TGFβ may act as ‘stop and go signals
to modulate postnatal ocular growth’ (Rohrer and
Stell, 1994). They would also be expected to have
considerable effects on the rate of lens growth and on
the expression of various genes in the lens (de longh
and McAvoy, 1992; Rohrer and Stell, 1994; Schulz et
al., 1996). Although not all sources of these factors
have been identified, the retina is a major contributor
(de longh and McAvoy, 1992). Light stimulation of
the retina would, therefore, be very likely to affect
their concentrations and subsequently impact on the
lens.
In the present study, this possibility has been
0014–4835}98}05066906 $25.00}0}ey980468 # 1998 Academic Press Limited
670 R. C. AUGUSTEYN
explored by examining the effect of light deprivation
on lens weight, protein content and crystallin dis-
tribution patterns in the mouse. It was observed that
lens growth was significantly slowed in the dark but
protein patterns and contents were unaffected.
2. Materials and Methods
All experiments with animals adhered to the ARVO
Statement for the Use of Animals in Ophthalmic and
Vision Research. Balb C mice were bred, reared and
maintained for up to 7 months in complete darkness in
the inner room of a modified constant temperature
(20°C) facility located in a basement. Entry to the
holding room was through a light proof annexe. The
room and annexe both lacked windows and each was
sealed with a heavy light proof door. All lights were
disabled. Access to the animals for cleaning, feeding
and sampling was restricted to the evening and no
lights were used in the vicinity of the holding room.
The mice were allowed unrestricted access to food and
water. Control mice were bred at the same time and
maintained up to 1 year under similar conditions
except that they were exposed to natural light}dark
cycles.
At various time intervals, light and dark reared mice
were killed by CO#asphyxiation and their body weights
and ages were recorded. The lenses were removed by
dissection, carefully freed of adhering tissue such as
vitreous and pigment epithelium, gently blotted dry
and weighed. Individual lenses were then homo-
genized in 1 ml phosphate buffered saline (PBS; 0±15
NaCl in 0±02 phosphate buffer, pH 7±2) and the
extracts were centrifuged at room temperature for
10 min at 11000 g to separate soluble and insoluble
constituents. Protein contents of the soluble and
insoluble fractions were determined as described
previously using the Lowry Method (Pierscionek and
Augusteyn, 1988).
The α-, β- and γ-crystallins were fractionated by
HPLC gel permeation chromatography at 20°C using
a Superose 12 column (Pharmacia) eluted with PBS at
1±0 ml min−". The weight proportions of the proteins
were calculated from the absorbance under the peaks
using extinction coefficients (Σ"
%,"cm) of 8±3, 22 and
24, at 280 nm, for the α-, β- and γ-crystallins,
respectively (Thomson and Augusteyn, 1985). At
least two lens extracts from different animals were
fractionated for each age and the results were
averaged.
The identities of the HPLC peaks were confirmed by
examining polypeptide patterns using sodium dodecyl
sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) for size distributions and isoelectric focusing
(IEF) in the range pH 4–9 for distribution of charged
species (Pierscionek and Augusteyn, 1988). The
proportions of crystallin polypeptides in the insoluble
fractions were determined by densitometric scanning
of the gels. Calculation of the total crystallin contents
from the insoluble and soluble protein data has been
described by Pierscionek and Augusteyn (1988).
3. Results
About 100 Balb c mice were bred in the dark and
maintained under the same conditions for various
times up to 7 months. Great care was taken to ensure
that the animals were at no time exposed to light.
They were housed in a disused constant temperature
facility which featured a small annexe between the
holding room and the outside and heavy light proof
doors into each room. The facility was in the already
dark basement of the building and all animal main-
tenance was carried out at night with no lights on
anywhere in the basement and both light proof doors
closed.
The mice reared in the dark were compared with a
similar number of age matched control mice which
had been maintained under natural light}dark cycles.
Body weight, lens wet weight, soluble and insoluble
protein contents and the proportions of α-, β- and γ-
crystallins were determined.
The relationship between body weight and age for
the dark and light reared mice is shown in Fig. 1. Most
growth took place within the first 50 days for both sets
of animals and continued at a gradually reducing rate
for the rest of the test period. The weights are
F. 1. Variations with age in body weight and lens weightfor Balb c mice raised in the dark (E) or in the light (D).
LIGHT AND LENS GROWTH 671
F. 2. Changes in lens proportion of body weight as afunction of body weight for Balb c mice raised in the dark(E) or in the light (D). In order to reduce the complexity ofthe figure, data were sorted into groups according to bodyweight and the averages were plotted. Each point shownrepresents the average of about 10 samples.
F. 3. The effect of age on the average proteinconcentration in lenses from Balb c mice raised in the dark(E) or in the light (D). Each point represents the average ofat least 5 different lenses.
surprisingly variable, given the genetic homogeneity
of the mice and the constancy of their environments.
For litter mates, body weights differed by as much as
30%. With increasing age, body weight became less
variable in the light exposed animals. Despite the
variability, it is clear that there are no significant
differences in body weight between animals reared in
the dark and light.
Lens wet weights also increased over the whole of
the period examined (Fig. 1). There are indications that,
at any age, lens weights are greater in the light reared
animals but this is difficult to gauge because of the
variability in the data. This may be a consequence of
the variability of body weights. Indeed, when lens
weight was expressed as a proportion of body weights,
the variability was markedly reduced and it became
obvious that lenses from animals reared in the dark
are smaller. The relationship is shown in Fig. 2.
Regression analysis confirmed that lenses from light
reared animals were a constant 0±031³0±004% of
body weight (310³40 µg g−", R#¯0±90) regardless
of age or body size. However, lenses from dark reared
animals represent progressively lower proportions
with increasing body weight, ranging from 0±031% at
birth to around 0±02% at body weights over 30 g. The
data in Fig. 2 suggest that the ratio will level out at a
value close to the 0±02%. The difference between the
light and dark reared animals was highly significant
(P!0±001).
Total protein contents were determined for the
lenses and used to calculate average lens concen-
trations. These are presented in Fig. 3. Although the
data are scattered, it is clear that there are no
significant differences between the light and dark
reared animals. Average protein concentrations for
the whole lens appeared to range from around
250 mg ml−" immediately after birth to about
700 mg ml−" in animals over 6 months old ("30 g
body weight). Insoluble protein contents were also
indistinguishable. In both sets of animals, insoluble
protein increased steadily from round 1% of the total
lens proteins at birth to 20% after 6 months (Fig. 4).
Isoelectric focussing and SDS-PAGE (results not
shown) revealed that the predominant constituents of
the insoluble fraction at birth were membrane and
cytoskeleton polypeptides (40–100 kDa). In the older
lenses α- and β-crystallins predominated. The patterns
were the same for light and dark reared animals.
In order to determine whether the patterns of
crystallin synthesis were affected by light exposure,
the soluble proteins in lens extracts were fractionated
on the basis of size, using HPLC gel permeation
chromatography. Identical profiles were obtained for
all mice of the same age, regardless of whether they
were raised in the dark or light, indicating that their
crystallin contents were the same. However, com-
parison of the patterns from mice of different ages
suggested that the proportions of the three crystallins
change with age in much the same way as has been
reported for bovine lenses (Pierscionek and Augusteyn,
1988). The weight proportions of the crystallins were
estimated from the areas under the peaks using their
extinction coefficients. The data are summarized in
Fig. 4 together with the insoluble protein contents.
At birth, the major constituent is α-crystallin which
represents about 46% of the total mass of lens protein.
As the animal ages, the soluble concentration of this
protein decreases slowly due to its insolubilization (Fig.
4) but the total remains constant. The β-crystallins
account for 17% of the total mass of protein at birth
and increase thereafter, with the soluble form reaching
23% at 6 months (Fig. 4). Substantial amounts of
insoluble β-crystallins (up to 9% of the total protein)
are also observed in the older lenses (results not
shown). The reverse is seen with the γ-crystallins
which represent 36% of the proteins at birth and
21% at 6 months (Fig. 4). However, SDS gels revealed
672 R. C. AUGUSTEYN
F. 4. The effect of age on the weight proportions ofcrystallins in lenses from Balb c mice raised in the dark (E)or in the light (D). Each point represents the average of atleast 3 different lenses.
that the γ-crystallin HPLC peak from the 195 day
sample contains about 10% β-crystallins. Thus, the
proportion of γ-crystallins is actually around 19%.
Very little of these proteins was observed in the
insoluble fraction, indicating that most of the decrease
is due to a reduction in their synthesis rather than
insolubilization. Mathematical modelling of the data
presented in Fig. 4, indicated that γ-crystallins
accounted for less than 7% of new proteins being
added to the lens after 6 months. These changes
resemble those seen in the bovine and human lens
(Thomson and Augusteyn, 1985; Pierscionek and
Augusteyn, 1988) but the extent of protein insolu-
bilization is greatest in the mouse.
It is clear from the data in Fig. 4, that there are no
substantial differences between the patterns of poly-
peptides synthesized in the lenses of mice reared in the
light or dark.
4. Discussion
The present study arose from our observations that
there are substantial changes in the patterns of protein
synthesis in human and bovine lenses around the time
of birth (Thomson and Augusteyn, 1985; Pierscionek
and Augusteyn, 1988). We had suggested that the
trigger for this switch might be the first exposure of the
retina to light. If this hypothesis were correct, light
deprivation would be expected to inhibit the alter-
ations in protein synthesis so that foetal synthesis
patterns would persist postnatally. Since testing this
hypothesis with human or bovine subjects is obviously
not feasible, we have used laboratory mice. This is not
an ideal alternative since the physical character-
istics—shape, size and hardness—of the lenses are
very different from those of human and bovine lenses.
However, the crystallin distributions and properties
are similar (Thomson and Augusteyn, 1985; Piers-
cionek and Augusteyn, 1988). It was not known if
there are changes around the time of birth but the
overall proportion of γ-crystallin in the mouse lens
decreases throughout life, as it does in human and
bovine lenses.
A comparison was made between the lenses of Balb
c mice maintained in the dark for up to 7 months with
those from animals kept under natural light}dark
cycles. Lens growth continued throughout the whole
of the study period and followed body growth in mice
maintained under natural light}dark cycles. In these
animals, lens weight represented a constant pro-
portion (0±031%) of total body weight regardless of
age or body size. By contrast, lens growth slowed in
the dark reared animals. At birth, the lens was again
0±031% of body weight but decreased to less than
0±02% after 6 months in the dark. Since the mouse
lens is essentially spherical, its radius may be calcu-
lated from the volume which can be estimated from
the wet weight and protein content. Lens radius in the
6 month old control mouse was 1±21 mm. In the dark
reared animal it was significantly smaller at only
1±06 mm (P!0±001).
Thus, our observations suggest that lens growth in
the mouse is sensitive to factors released in response to
light. These factors are required continuously since
lens growth continues throughout life, albeit at a
gradually declining rate. Lack of these factors results
LIGHT AND LENS GROWTH 673
in a slower rate of growth. Since the lens does not
appear to possess receptors for visible light, one can
only surmise that the signals emanate from the retina
which has an abundance of photoreceptors. The most
likely substances causing the lens stimulation are the
Fibroblast Growth Factors, particularly the bFGF (FGF-
2), which have been shown to affect the rate of
epithelial cell proliferation, migration and differen-
tiation (de longh and McAvoy, 1992). The Trans-
forming Growth Factor beta (TGFβ) which attenuates
the effects of other growth factors may also play a role.
Large amounts of the inactive precursor for this Factor
are present in the vitreous.
The decrease in lens size in the mouse appears to
differ from the responses observed with other species in
which visual deprivation leads to ocular elongation
while the lens is not affected (Mutti et al., 1996).
However, such observations have generally been made
over the short times needed to induce myopia, typically
7–14 days in chicks. It may be that lens changes are
too small to detect in that time. Our data reveal that
mouse lens thickness was reduced by only 5% after 1
month in the dark. It has also been reported that
constant light exposure results in a decrease in lens
thickness (Stone, Desai and Capegart, 1994). This
suggests that light, per se, may not be sufficient for
optimum lens growth. It would appear that variations
in visual stimuli are required.
In the absence of any other ocular changes, the
observed decrease in lens size would result in greater
lens power and myopia. It is probable that eye length
will also have increased, as has been observed in other
species following light deprivation, thereby exacer-
bating the myopia (Mutti et al. 1996). With hindsight,
it is unfortunate that ocular dimensions were not
determined but, this study was undertaken for a
different purpose, to examine biochemical changes.
Age related changes in the proportions of the
crystallins were observed in both sets of animals. It
would appear that α-crystallin represents a constant
46% of the total mouse lens proteins throughout life
but some of the protein becomes insoluble as the lens
ages. This may be due to an increase in molecular
mass such as that observed in human (Thomson and
Augusteyn, 1985) and bovine (Pierscionek and Augus-
teyn, 1988) lenses. The available data suggest that α-
crystallin may provide a constant background of
protein properties which are important for proper lens
functioning at all stages of development and growth.
One such property may be the chaperone activity
which is generally believed to play a role in main-
taining lens transparency by preventing the pre-
cipitation of denatured proteins (Horwitz, 1992).
By contrast, the levels of the other proteins vary.
The β-crystallins increase from around 17% at birth to
32% at 6 months. Some of these also become insoluble
as the lens ages. This increase is matched by a decrease
in the γ-crystallins from 36% to 19%. Similar
replacement of γ-crystallins by β-crystallins has been
observed in other species (Jobling, Stevens and
Augusteyn, 1995; Pierscionek and Augusteyn, 1988;
Thomson and Augusteyn, 1985) and was the stimulus
for the present study. Mathematical modelling of the
data indicates that the changes are due to a greater
than two fold increase in the proportion of β-crystallin
synthesis and an over 80% reduction in γ-crystallin
synthesis in the adult lens cortex. The significance of
this replacement has not been established but it may
be responsible for the formation of concentration
(hydration) gradients in the tissue. High levels of γ-
crystallins seem to promote the formation of hard,
dehydrated tissue such as that found in the nuclear
region of most mammalian lenses, whereas β-crystal-
lins are usually associated with the softer more
hydrated tissue.
No differences could be detected between the light
and dark reared animals in total lens protein contents,
concentrations or in the proportions of soluble and
insoluble proteins at any given age. In addition,
polypeptide distributions were identical in light and
dark reared animals. Thus there appear to be no
differential effects on gene expression which can be
attributed to light exposure or deprivation. Perhaps,
the changes in lens protein synthesis patterns are due
to factors such as the levels of hormones and other
cytokines, specifically associated with parturition.
Further studies will be undertaken to examine this
possibility.
Acknowledgements
This work was supported in part through the Ocular Lensproject of the Cooperative Research Centre for Eye Researchand Technology (CRCERT).
Nigel Parker and Zhiping Fang provided valuable as-sistance in parts of this study.
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