the effect of light deprivation on the mouse lens

6
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 for the 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, reared and maintained in complete darkness for up to 7 months. Data were collected on body weight, lens weight, lens protein contents and crystallin distributions. The data were compared with those obtained from age matched mice maintained in natural light}dark conditions. No significant differences were observed in body weight between animals maintained in the light and dark. However, animals kept in the dark had significantly smaller lenses. After 6 months in the dark, the lens represented 002% of body weight compared with 0031% in the light reared animals (P ! 0001). Lens protein concentration, insoluble protein contents and crystallin synthesis patterns were indistinguishable for the two groups of animals. It is concluded that light stimulation of the eye is required for optimal lens growth but does not affect the 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 γ s (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 Cardigan Street, 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

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Page 1: The Effect of Light Deprivation on the Mouse Lens

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

Page 2: The Effect of Light Deprivation on the Mouse Lens

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).

Page 3: The Effect of Light Deprivation on the Mouse Lens

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

Page 4: The Effect of Light Deprivation on the Mouse Lens

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

Page 5: The Effect of Light Deprivation on the Mouse Lens

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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