Eye lens ageing in the dogfish (Mustelus canis)

Comp. Biochera. Physiol., Vol. 63B, pp. 379 to 385

0305-0491/79/0107 0379502.00/0

© Pergamon Press Ltd 1979. Printed in Great Britain

EYE LENS AGEING IN THE DOGFISH

(MUSTELUS CANIS) SEYMOUR ZIGMAN and TERESA YULO Ophthalmology Research Laboratory, Department of Ophthalmology, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue (Box 314), Rochester, New York 14642, U.S.A. and The Marine Biological Laboratory, Woods Hole, Massuchusetts, U.S.A. (Received 1 December 1978)

A~tract--1. Age-related alterations in the distribution of water-soluble, high molecular weight (colloidal), and water-insoluble proteins of the lens of smooth dogfish (Mustelus canis) were measured. 2. The ages of these animals ranged approx from 2 to 50 yr, during which time the lenses grew from 100 to 1500 mg (wet wt). The lenses contained approx 50~o water. 3. Water-insoluble protein accumulated to a level greater than 50~o of the total proteins by the time the animals reached maturity. The lenses of other animals, such as mammals and humans, would be opaque if they had a similar insoluble protein content. 4. Each protein fraction contained the same protein chains (mol. et 1900-25,000 daltons), as observed by SDS polyacrylamide gel electrophoresis, except the water-insoluble fraction, which seemed to contain several extra protein chains with higher molecular weights, which represent fiber cell membrane components. 5. Further purification of these fiber cell membranes indicated that their protein chain makeup was mainly from the same low molecular weight chains present in the soluble and high molecular weight colloidal proteins. INTRODUCTION The physical appearance of the eye lens, its growth process, and the molecular weight of the peptide chains of its structural proteins do .not vary appreciably among all vertebrates. However, accommodation is accomplished in two quite different ways. In humans, the disc-shaped lens is stretched to become thinner and relaxed to become thicker for far and near vision, respectively. In dogfish, the spherical lens is moved more anteriorly to view closer and more posteriorly to view distant images. Thus, in evolution, there was a change from a spherical hard lens that is difficult to deform (i.e. dogfish) into a disciform elastic lens (i.e. human). Evolutionary aspects of dogfish lenses are discussed by Duke-Elder (1956). Since the subunit peptides of the lens crystallins found in both human and dogfish lenses are similar in size, the basis for the differences in the mechanical nature of the accommodation process must be dependent upon the manner in which these subunits are built into larger structures. One examination of the highly aggregated protein fractions of the dogfish lens showed that they are present at higher levels at an earlier age than in human lenses (Yulo & Zigman, 1977), and that the subunit peptides are much more firmly entrenched in the fiber cell membranes (Griess, et al., 1974). But the dogfish lenses are clear when the insoluble protein aggregates are present at levels much higher than those in human lenses, even in human lenses of advanced age, and most cataracts. This aggregation process may depend upon the matrix of elongated and thick fiber cell membranes to which the crystallins attach. Thus in both dogfish Supported by: Research Grant No. EY 00459 from The National Eye Institute of the National Institutes of Health. 379

(Lerman & Fontaine, 1962) and human lenses (Clark, Zigman & Lerman, 1968) there is an accumulation of so-called "water insoluble" or "albuminoid" proteins with ageing. This process would appear to continue in both species for their entire lifetimes. This commonication documents the time course of the growth and ageing of the dogfish lens, and the accumulation of proteins in the low molecular weight soluble phases, in the high molecular weight or colloidal phases, and in the insoluble phases which contains both firmly bound and loosely bound crystallins plus the fiber cell membranes. The latter fraction was further subdivided into the urea-solubilized and ureainsoluble portions and, finally, into the membraneenriched proteins themselves. MATERIALS AND METHODS The study was carried out using fresh or fresh-frozen lenses of Mustelus canis removed from animals of varying length, and lens size. Animal length varied from 10 to 50in., and lens weight ranged from 50 to 1500mg. These lenses were homogenized in pH 7.0, 0.1 M PO,,, 0.1 M EDTA buffer, and were separated by differential centrifugation at 12,000g to sediment the insoluble fraction and at 100,000g to sediment the high molecular weight colloidal fraction (see Zigman et al., 1976). The supernatant was designated as the soluble fraction. In some cases, the aqueous-insoluble protein fraction was further separated into 7 M urea-soluble and urea-insoluble fractions. In other cases, the urea-insoluble fraction was further washed exhaustively until no further protein was detected to be extracted. When this technique was done on unhomogenized material, it was observed microscopically to contain empty fiber cell membranes. All fractions were sulfonated as described previously (Zigman et al., 1976), and then were subjected to polyacrylamide gel electrophoresis in 0.1 M sodium dodecylsulfate (pH 7.0 in 0.1 M PO,,) to separate the subunit peptides.

380

SEYMOUR ZIGMAN and TERESA YULO

20

1.6

O0

~ o.8 d

00 ~ 00o0 O

0.4

0

I I0

0

,I/°" I 20

I 30

.,./ O@

I 40

I 50

I 60

Body length (in) Fig. 1. Lens growth (increase of wet weight, mg) as a function of dogfish age (increase of body length). Each point represents the average value of the weight of the two lenses of an animal. The conditions were: 7.5~{, of polyacrylamide in PO4, buffer, pH 7.0, 2 hr of time, at 90 ma. Gels were stained with 0.5% coomassie blue in 50"~, methanol and destained with 50% methanol and 7% acetic acid and then 7% acetic acid. The gels were then scanned using an E.C. Apparatus, Inc. densitometer to obtain the migration distances and the quantitative levels of each band observed.

RESULTS

The relationship between lens growth, as represented by its wet weight, and dogfish age, as represented by body length, is shown in Fig. 1. Although no exact method has been available to determine the chronological age of these animals, an estimate of the

6°° L 300

I S is w~ter soluble, low mol. wt. o C is colloidal intermediate mol. wt. ]' is water insoluble, high mol. wt.

® •

,o

20

~

IO

15

t

20

25

30

/

,

/

I

4O

,

~

C

I

45

I

50

Length of animal (in) Fig. 2. Changes in the levels of proteins present in the soluble, high molecular weight colloidal, and water-insoluble fractions as a function of dogfish age. See Methods and Materials for details of the preparation of these fractions. Each point is the average of three lenses.

Eye lens ageing in the dogfish (Mustelus canis)

381

age was made using data on a related species, Squalus ance of the fibers after successive extractions as outacanthius, published by Ketchen (1975). From this lined above. The SDS-PAGE analyses of the high molecular estimate, the animals of this study range approximately from 5 to 50 yr. The dry weight of the lenses weight (colloidal), of the urea-soluble and ureaof all age categories of this study was 50~ of the insoluble portions of the water-insoluble protein, and of the purified membrane preparations are shown in wet weight. Lens protein accumulation and distribution among Fig. 5. The distribution of the urea-soluble and ureathe fractions separated is illustrated in Fig. 2. While insoluble fractions has already been reported (Zigman the soluble proteins were present at about five times et al., 1970). While the predominant chains in all of the level of the insoluble proteins in the youngest ani- these fractions have molecular weights of 19,000mal lenses, there is an equivalence of these fractions 25,000 daltons, there are great similarities between the in the medium-aged animals, and approximately a distribution of the heavier chains comprising the high two-fold excess of insoluble proteins over the soluble molecular weight and urea-soluble electrophoretic proteins in the oldest lenses. The level of high molecu- profiles, and also between those of the urea-insoluble lar weight colloidal protein was fairly constant at and purified membrane preparations. The latter two about 3-10 mg per lens throughout the age process. fractions are highly enriched with two bands of 41,000 As shown by Griess et al., (1974), Sephadex G-150 and 52,000 daltons which may be the exclusive memgel filtration of dogfish soluble proteins reveals that brane protein subunits. The data also shows that the the majority of these undenatured proteins have mol- membranes always contain some 19,000-25,000 ecular weights ranging from 30,000 to 15,000 daltons, daltons peptides, no matter how thoroughly the a situation that is dissimilar to that in mammals, in membranes are washed or when they are extracted which a majority of the soluble protein species are by reagents that do not disrupt covalent bonds. of molecular weights greater than 30,000 daltons. When analyzed by SDS-PAGE analyses, the major DISCUSSION subunit sizes in all fractions of dogfish lens proteins also had molecular weights between 19,000-25,000 The results show that the ageing process of the dogdaltons, but in the high molecular weight colloidal fish lens involves the synthesis of low molecular and, especially in the water-insoluble fractions, weight protein species that interact and aggregate to greater subunit sizes were detected. Table 1 summar- form a preponderance of very high molecular weight izes the molecular weight of the subunits detected in species. It is important to note that this study covers the separated fractions of medium-sized dogfish a period of time of at least 50 yr duration, and is lenses. Figures 3(a, b, c) illustrate the densitometer not as short a period of time covered in most animal tracings of SDS-PAGE analyses of the protein frac- studies, which are generally only 2-3 yr. In this retions of dogfish lenses, respectively. Most striking is spect, a time duration similar to that in humans is the appearance of numerous b/~nds heavier than the represented. 19,000-25,000 daltons, which results most strikingly Another striking finding is that the fraction with with increasing animal age. This result is emphasized the highest molecular weight species accumulates by Fig. 3, which shows the accumulation of heavy much earlier and reaches a higher level in the normal peptide chains when the soluble, high molecular dogfish lens than in normal human or other mamweight, and insoluble fractions are compared in malian lenses. Human lenses in which the insoluble protein level is greater than 40To of the total protein young and old dogfish lenses. Attempts were made to obtain fiber cell membranes as observed by Zigman, et al., (1976), are all cloudy that were free of loosely bound lens crystallins. It was and are called cataractous lenses. A question raised found that intact lens fiber membranes could be pre- but not answered here is what factors in the lens pared by teasing and soaking lenses in elasmobranch allow the tissue to retain its transparency in spite of Ringer's solutions and then extracting the lens tissue the presence of such high levels of large aggregates obtained with distilled water first, and then with 8 M which should be scattering much of the incident light. urea. Micrographs of this process are shown in Since the dogfish lens is relatively dehydrated (at 50To Fig. 4, which demonstrates the changes in the appear- protein compared to only 30% protein in human lenses), this may be one factor involved. The high tissue urea level (0.3 M) may be another. It has been shown that while this is a low concentration of urea Table 1. Peptide chain sizes in dogfish* lens to influence protein chain interactions, it is sufficient to interfere with the cold cataract or protein cryopreProtein fractions (~o) cipitation of the dogfish lens (Zigman & Lerman, High 1964). molecular Mol. wt weight Even though accommodation is accomplished difFractions (daltons) Soluble (colloidal) Insoluble ferently in dogfish than in mammalian lenses, the ageing process is quite similar in producing high molecu1 63,000 0.6 1.I 3.6 lar weight aggregates. Both the internal and external 2 52,000 1.7 2.8 9.7 environment of the dogfish and mammalian or 3 41,000 2.6 6.4 12.2 human lens are drastically different. For example, 4 25,000 5 19,000 ) 91.7) 82.6) 67.1 dogfish eye fluid tonicity is much greater than that of humans, and it contains urea at about 0.3 M for 6 13,000 3.6 7.4 7.4 osmotic balance against the seawater. Also, the dog* 45 in. body length, i.e. mature animal. fish lens normally is not exposed to sunlight or to

382

SEYMOUR ZIGMAN and

~.~_~ ~.~

TERESA Y U L O

Z

~

.~-~g ~ .~ -=._ ~'~

~~ =~

--

,1~ ~

._~N

~

-r

~

~-

e,l

~

o

o

o

o

~D

T

~.l!sue[~

o~ ~

o

~

!

I

~

~

,~l.!~u~ ~ o

~

o

o,

~

C | .~ _~ B ~ -®~ k ~ ._~ #:~ ._~~

E=

&~,o r

~T~

J

J

~E ~

o

! • 1" _:

I

C~

T

•

0

T

"

aO

LO

~-

0

0

0

~

~

~

o

~8

~

C~

I

~

"~ o S ~, ~

~!$ueO 0

'

~

~'

;

o

o

t

i~

~o~

~

o o

.~

~

Eye lens ageing in the dogfish (Mustelus canis)

383

t6 Insolubleproteins~..~ 3r' animal f '1.4

Insolubleproteins 27" animal

1.2 0

;).8 i

}6 D.4

).2 D I

I

I

2.5

I

I

I

0

5.0

Insolubleproteins 41" anim~v//~

I

I

2.5

I

I l 5¸0

I

I

Insoluble 46" animaI

~6

14 12 qO

~

Q8 i

.(1.6 0.4 02 0

I

0

2.5

5.0

I

0

~

I

I I 2.5

I

1

5.0

~i~o~ionin ~1 ~c~ Fig. 3(c) Fig. 3. Densitometric tracings of the SDS-polyacrylamide gel electrophoretic profiles of dogfish lens protein fractions as a function of age. (a) Water-soluble proteins; (b) high molecular weight (colloidal) proteins; (c) water-insoluble protein. Conditions of protein preparation and treatment for the electrophoresis, and conditions of electrophoresis are given in "Methods".

{b)

[c)

Fig. 4. Photomicrographs of lens fibers prepared by teasing and soaking in elasmobranch Ringer's solutions rather than by homogenization, and appearance of the fibers after subsequent extractions. (aj No extractions: {bl water-soluble material removed: (el urea-soluble material removed. Other procedures used are given in the Methods.

[a)

>

> Z

385

Eye lens ageing in the dogfish Mustelus cams) (o)

Ureo soluble

(b)

°.7'27'"

~

HM colloidal ]~ / ~

16, 1,4

1.2. I0. 0.8 0.6 0~.

1

0

1

I

4,5,000 25,000 14,000 MoSecular weigt (daltons)

I

0

~

I

45,O00 25,000

Molecdar ~ N

I

~4,000

1~1

Fig. 5. Densitometric tracings of the SDS-polyacrylamide gel electrophoresis profiles of the aggregated protein fractions of dogfish lenses. (a) High molecular weight (colloidal) and urea-soluble (waterinsoluble) fractions; (b) urea-insoluble and membrane fractions. Details of the procedures are given in the Methods. the non-aquatic environment as are mammals and humans, and its body temperature is always at least 20°C, or lower. These considerations seem to indicate that the ageing of the proteins of the lens in the sense of aggregation has a unique mechanism that is at least partly independent of internal and external environmental conditions. We also conclude that the dogfish lens is a good model for studies that retiect lens ageing mechanisms with similar molecular ageing parameters as in mammals, but devoid of terrestrial environmental condititions. A final consideration to be made at this time is that the lens fiber cell membranes are composed of only small numbers and amounts of peptide chains other than the soluble crystallins themselves. This indicates that the membranes consist of a small amount of certain specific membrane proteins and a large amount of covalently associated lens crystallin. In this instance, it may be that crystallins comprise the membrane itself, and that the aggregation process is related to a seeding of the crystallins by the fiber cell membranes. REFERENCES

CLARK R., ZlGMAN S. ~g~LERMANS. (1969) Studies on the

structural proteins of the human lens. Expl Eye Res. g, 172-82. DCKE-ELDIm W. S. (1958) The Eye in Evolution. System of Ophthalmology, Vol. 1. C. V. Mosby, St Louis, Mo. GRmss G., Z]GU^N S. & YULOT. (1974) Formation of dogfish lens fibers from soluble crystallins. Biol. Bull. 147(2), 478-479. KETCH~N K. S. (1975) Age and growth of dogfish Squalus acanthias in British Columbia Waters. J. Fish Res. Bd Can. 32, 43-58. LE~UAN S. & FONTA]NEJ. (1962) The effect of ageing on protein and RNA metabolism in the dogfish lens. Growth 26, 111-116. To,AN N., Z]GUAN S. & FA~NSWO~Tn P. (1978) In preparation. ZmUAS S., Mc~r~o J. & LE~UAr~ S. (1965) Effect of urea on the cold precipitation of protein in the lens of the dogfish. Nature, Lond. 2117, 414-415. ZmUAN S., ScrrULTZ J. & YCLO T. (1970) Variations in the makeup of lens insoluble proteins. Expl Eye Res. 10, 58-63. Z~GUA~ S., G~orr J., YCLO T. & GRmss G. (1976) Light extinction and protein in lens. Expl Eye Res. 23, 555-567. ZmUAN S. & YCLO T. (1978) Ageing and cataract in dogfish (Mustelus canis) eye lenses. Biol. Bull. In press.