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A light microscope study of the lens of the tryptophan deficient rat
E.rp. Eye RPS.(1979) 28, 533-538
A Light Microscope Study of the Lens of the Tryptophan Deficient Rat
The lens of the rat, fed on a tr!-ptophxIl.tleficieilt diet is smaller than normal and many of the aortieal fibre cells have abnormal morphologies. These cells are probably related to. if not the direct cause of. the inner reflecting surface wen within the lens when the eye is Cewcd in the slit lamp. The srquence of changes in crystallin synthrsis during the formation of lens fibre ~11s in the ne\\ born tryptophan-deficient) rat was examined by immnnofluorescence. There were no differences between the normal and the tryptophan-deficient rat in the distribution of x-,/3 - and y-crystallins. Therefore, in this respect at least, the differentiation of lens fibres ill newborn tryptophan-deficient animals was normal. lens tihres: rrystallins: lens size: immunofluorescence. Kr!/ wov1.u: tr3ptoI)lian-deficiellcv:
1. Introduction Eq~erimental cataract caused by dietary deficiencies is rare. The only authenticated and repro(lucible example is that produced in guinea pigs and rats by feeding a diet cleficient, or totallr lacking in the essent’ial amino-acid, tiyptophan (Totter and Day, 1942 ; van Sallmann, Reid. Grimes and Collins. 1959; Dische. Elliott, Pearson and Merriam, 1959; Bunce and Hess, 1956: ran Heyningen. 1976; Ohrloff et al., 1958; 13unce. Hess and Fillnow, 1978). Hist’ological studies of lerises of rats fed on a Drypto1)hawdeficient diet have revealed decomposition of lens fibre cytoplasm at the anterior sutures (van Sallmnnn et al.. 1959) and changes in the numberz distribution and nlorphology of nuclei in the lens bow (McAvo:, and van Heyningen. 1976). Other st,utlirs have shoxn a reduction in the rate of weight increase and in the proportion of x~htl& lens proteins (Dische et, al.. 1959; ran Heyningen. 1976; Ohrloff et’ al.. 1978: Buncc et al., 1978). This study shows that lenses from tryptophan-deficient rats are smaller t,han lenses frown controls. In these smaller lenses many of the cortical fibre cells have al)normal morphologies. One explanation for these observations is that tryptophanTo examine this possibility. the deficiency causes abnormal fibre differentiation. clistributions of three major groups of water soluble lens proteins, x-. p- and ycrystallins (see Clayton, 1974; Harding and Dilley, 1976 for reviews). were established in newborn rats of trpptophan-deficient and normal mothers by immunofluorescence. 2. Materials and Methods The tryptophan-deficient diet was based on that used by van Sallnlanu et al. (1959) the calculated L-tryptophan content was reduced to @7Oq/,.The diet contained soya
but
* Present address: Department of Histology 2006, New South Wales, Aust’ralia. 0014-4835/79/050533+06 c
and Embryology,
$01.00/O
The University
of Sydney, Sydney
0 1979 Academic Press Inc. (London) Limited 533
A34
,J. 11’. MrAi’OY,
L. ,J. PALFREY
ASD
R. VAN
HEYNINGEK
bean protein 4 :,A, gelatin 4%. casein acid hytlrolysate S’$&, corn meal 32q,, corn oil 10”~. sucrose 35.2yb, salts 6’$/,. u,L-methionine 0.4%, u,L-lpsine 0.2~/0, inositol O.l’:;, cliolitle chloride O-l;/, and vitamins. Litter mates of Wistar strain albino rats were used. Ha,lf were weaned onto the deficient diet alone and the other half onto the deficient diet, with L-tryptophan (2 g/kg). Food and water were given ad lib. After 4-5 weeks on the diet the rats were killed and the eyes prepared for histology. In another experiment female rats were placed on the deficient diet, and after 2 weeks were allowed to breed. The females that conceived were maintained on the diet. The newborn rats were killed and the eyes prepared for histology.
Eyes were fixed in Carnoy’s (3: 1 ethanol : acetic acid) for 30 min and ettlbetldetl in paraffin. Serial sagittal sections were cut at 5 ~111.Xea,surements and volume calculations were made from three sections from the middle of the lens (the largest sections). These were made according to method II of Wolpow and Marimont (1967) which assumes that t’lle lens is an ohlate spheroid. Measurements xnd then volume calculabions were made for each of the three sections. The average of these was taken as the lens volume. The volumes of five experimental and three control lenses from rats 4-5 weeks on the trvptophall-deficient diet as well aq volumes of four experimental and four control ne~~~.hortt rat lenses were c,alculat,ed.
Anti-crystallin antibodies prepared as described previously (McAvo~, 1978) were used to localize x-, /3- and y-crystallins in sections of eyes by the indirect, 1n~nlu~lofluoreacelIt technique of Weller and Coons (1954). Whole eyes and in some cases lens segments were fixed in Carnoy’s at room temperature for 30 min, routinely prccessed aud embedded in paraffin. Antibodies were applied to 4 pm sect.ions followed by goat anti-rabbit, gamma globulin antibody conjugated with fluorescein isothiocyanate (FITC, Nordic Imn~unological Laboratories, The Netherlands). To determine the specificity of t’he fluorescence the following two controls were carried out. Serum from non-immunized rabbit* was substituted for specific anti-crystallin antisera. The other control was to apply FITC directly to the section Gthout prior incubation it1 serum. In both controls no fluorescence \txs obser\-ed.
3. Results and Discussion
Lens volumes of tryptophan-deficient and control rats were calculated front sagittal s&ions. The volumes of two age groups of rats were calculated; newborn rats whose mothers were fed on the trpptophan-deficient diet for 2 weeks before conception anal rats fed on t#he diet from 4-5 weeks after weaning. The results are shown in Tat& T. In both experime.nts the lenses from tryptophan-deficient rats arc sttlaller than lenses froltl control animals. These volume differences bet,ween tryI’tol)ll~~tl-tleficient and controls are in general agreement with lerls weight differences (L)isch~ et al.. 1959; van Heyningen, 1976; Ohrloff et al.. 1978; Runce et al., 197X). Tbs. in t,oth c,xperiments reduced tryptophan intake resulted in slower than normal lctls gron-th.
We previously reported that. in tryptophan-deficient deeper into the lens and are 2- 3 times more numerous
rats, nucIeatec1 cells extend than in controls. In addition
(‘HASGES
IN
THE
TRYPTOPHAN-UEFIC’IENT
LESS
35
TABLE I
Lens volumes of tryptophan-dejicient and control rats of two age groups (stdard deviations included) Rats
Lens volume cont~rols
Newborn 1-5 weeks post weaning
I~S~W4 1’.7& 1.0
(mm”)
Tryptophan-deficient
1.0*0.5 8.6&0.9
nuclei with abnormal morphologies are present (McAvoy and van Heyningen, 1976). In the study reported here the cells with the abnormal nuclei are shown to have a variety of shapes and in general are much thicker than normal fibre cells (Fig. 1). In both anterior and posterior lens cortex there is an abundance of cells with abnormally large diameters. Also in some cases there are what appear to be areas of fibre cell decomposition. The cells have variable diameters and do not form regular rows as do the fibre cells in normal lenses (Fig. 2). They are probably the anterior and posterior ends of the large cells with abnormal nuclei. These large cells might be swollen fibre cells or the result of the fusion and decomposition of &re cells. Indeed, swelling may precede fibre fusion and decomposition.
F’rc:. I. (a) The lrns bow of a rat fed on the tryptophan-deficient diet for 5 weeks postweaning. Haematosylin. phase contrast, Y 120. (b) This urca is indicated on (a) and shows detail of the fibrt ccllx with ;tl~nwmal morphologies (arrow). Havmatnxylin, phase contrast, ,: 480.
FIG. 2. Anterior lens cortex of tryptophan-deficient and control rats, 5 weeks postweaning. Haomatoxylin, phase contrast, > 130. (a) Control rat. The diameters of fibre cells do not, vary to the extent shown in (b) and (c). Fibre cells are regularly packed into rows. (b) Tryptophan-deficient rat. Tho diameters of the fibre cells are abnormally large and very variable in size. (c) Tryptophan-deficient rat. Thr amorphous area (arrow) may represent, a region of fibre cell decomposition.
These ahxlornlalities are probably related to, if not the direct cause of. the inner reflecting surface within the lens as seen when these tryptophan-deficient rat eyes are viewed in the slit lamp (see van Heyningen. 1976).
Distribution
of qstallins
in the tryptophnn-dejcielzt
mt Lens
It was found that in thenormal 4-5-week-old rat lenses the localization of’ crystallins by inmumofluorescence gave inconsistent results. The main observatiom were that
(‘HAXGES
IN
THE
TRYPTOPHAN-DEFICIENT
LENS
537
after sections were treated with crystallin specific antibodies and FTTC a variable number of the superficial lens fibres fluoresced and the deeper lens fibres did not fluoresce. This was the case when all three groups of antibodies were used. The absence of fluorescence after antibody treatment has also been observed in central fibres of mature chick lenses (Ikeda and Zwaan, 1967 ; Rrahnia and vau Dooreuuiaalen. 1971). For this reasou a meaningful comparison between tryptophan-tlcficiellt, ant1 control lenses front this age group could not be made. The localization of crystallins in newborn rat lenses gave consistent results. Sagittixl sectiom of tryptophau deficient lenses treated with anti K-MltihJdieS showed apple ~reea fluorescence in all lens cells [Fig. 3(a)].
ITsiug mti P-antibodies the green fluorescence was not tlet’ected in all lens ~11~;. /j-(‘rystallirl was detected only iu the cytoplasm of fibre cells and in cells at t’he icus equator that hat1 just begun to elongate [Fig. 3(b)]. I!sing anti y-antibodies green fluorescence was onlv found in the cytoplz,sm of fitne cells. In this respect the distribution of y-was similar to /3- but different in that it was first clet,ect,ed in cells that had considerably elongated [Fig. 3(c)]. The tlistributions of the three major groups of crystallins in the smaller lenses of tryptophan deficient rats are essentially similar to that previously described in lenses from normal new-born rats (Mcdvoy, 1978). Thus, in newborn animals at least, try@olhan deficiency does not alter t’he sequence of changes in crystallin synthesis tlurin~ the formation of fibre cells. ACKNOWLEDGMENTS
The support of this research was provided by grant no. 1 ROl EY 01548-01 awarded by the N.E.I. to Ruth van Heyningen.
5x3
J. W. McAVOY,
L. J. PALFREY
AND
K. VAN
HEYKINCEX
REFERESCES Brahms, S. K. and van Doorenmaalen, I$:‘. J. (1971). llllrnnnofluoresuence studies of chick lens FlSC and sc-crystallin antigens during lens morphogenexis and development. Ophthtrl. fi’~s. 2, 344-67. of Bunce, G. E. and Hess. J. L. (1976). Lent,icular opacities in young rats as a ~OINY~LIHII~A maternal diets lo\\. in tryptophan and/or Vit,amin E. J. AVufr. 106, 222-g. Brmce, G. E.. Hess, J. L. and Fillnow. G. M. (1978). Investigat,ion of low tryptophan intlucrtl c*at,aract in weanling rats. Ezp. Eye Res. 26, 399-405. Clayton. R. 31. (1974). Comparative aspects of lens proteins. In ‘I’he Eye, Vol. 5 (&Is Davson. H. and Gmham, L. T.). Pp. 399-494. London: Academic Press. Dische, Z., Elliott, J.. Pearson, E. and Merriam. (:. IX. (1959). Changes in proteins and protriu synthesis: In tryptophane deficiency and radiation cataracts of rats. dm. J. Ophthrtl. 47, 36X-79. Harding, J. ,I. and Dilley, K. J. (1976). Sitructural proteins of the mammalian lens: a revie\\ with emphasis on changes in development, ageing and cat’aract. E’zp. Eye Res. 22, l-73. Ikeda. A. and Zwaan, J. (1967). The changing celiular localization of n-crystallin in the lens ot the chicken embryo, studied by immunofluorescence. Ded. Biol. 15, 348-67. Xcdvoy, J. FT. and van Heyningen, R. (1976). Changes in the cells of the lens bow and epitheliunl of tryptophan-deficient rats. Colloq. d’Iruernl 60, 245-50. MrAvoy, J. IV. (1978). Cell division, cell elongation and distribution of s(-, ,8- and y-orystallins in the rat lens. J. Ernbryol. Exp. Morph. 44, 149-65. Ohrloff, C., Stoffel, C., Koch, H.-R., Wefers, U.. Boars. J. and Hockwin, 0. (1978). Experimental cataracts in rats due to tryptophan-free diet. dbrecht. r. Gmefes. A4rch. Ophthnhrrol. 205, 73-9. Totter, J. R. and Day, P. L. (1942). Cataract and other ocular changes resulting from t’ryptophanc deficiency. J. AVutr. 24, 159-66. van Heyningen. R. (1976). Experimental studies on cataract. J~csf. Ophthdrnol. 15, 685-97. von Sallmann. L., Reid, RI. E., Grimes, P. -4. and (‘ollins, E. 81. (1959). Trpptophan-deficirn(,>cataract in guinea pigs. arch. Oph.thaZnzol. 62, (352 72. \Veller, T. H. and Coons, A. H. (1954). Fluorescent antibody studies with agents of ~~icellrc :lntl herpes zosfer propagated in vitro. Proc. Sot. E.rp. Viol. Jletl. 86, 789-94. \Volpow, E. 11. and Marimont, R. B. (1967). Mathematical approach to measurements on embryonic chick lens. J. Morph. 121, 71-80.
A Light Microscope Study of the Lens of the Tryptophan Deficient Rat
The lens of the rat, fed on a tr!-ptophxIl.tleficieilt diet is smaller than normal and many of the aortieal fibre cells have abnormal morphologies. These cells are probably related to. if not the direct cause of. the inner reflecting surface wen within the lens when the eye is Cewcd in the slit lamp. The srquence of changes in crystallin synthrsis during the formation of lens fibre ~11s in the ne\\ born tryptophan-deficient) rat was examined by immnnofluorescence. There were no differences between the normal and the tryptophan-deficient rat in the distribution of x-,/3 - and y-crystallins. Therefore, in this respect at least, the differentiation of lens fibres ill newborn tryptophan-deficient animals was normal. lens tihres: rrystallins: lens size: immunofluorescence. Kr!/ wov1.u: tr3ptoI)lian-deficiellcv:
1. Introduction Eq~erimental cataract caused by dietary deficiencies is rare. The only authenticated and repro(lucible example is that produced in guinea pigs and rats by feeding a diet cleficient, or totallr lacking in the essent’ial amino-acid, tiyptophan (Totter and Day, 1942 ; van Sallmann, Reid. Grimes and Collins. 1959; Dische. Elliott, Pearson and Merriam, 1959; Bunce and Hess, 1956: ran Heyningen. 1976; Ohrloff et al., 1958; 13unce. Hess and Fillnow, 1978). Hist’ological studies of lerises of rats fed on a Drypto1)hawdeficient diet have revealed decomposition of lens fibre cytoplasm at the anterior sutures (van Sallmnnn et al.. 1959) and changes in the numberz distribution and nlorphology of nuclei in the lens bow (McAvo:, and van Heyningen. 1976). Other st,utlirs have shoxn a reduction in the rate of weight increase and in the proportion of x~htl& lens proteins (Dische et, al.. 1959; ran Heyningen. 1976; Ohrloff et’ al.. 1978: Buncc et al., 1978). This study shows that lenses from tryptophan-deficient rats are smaller t,han lenses frown controls. In these smaller lenses many of the cortical fibre cells have al)normal morphologies. One explanation for these observations is that tryptophanTo examine this possibility. the deficiency causes abnormal fibre differentiation. clistributions of three major groups of water soluble lens proteins, x-. p- and ycrystallins (see Clayton, 1974; Harding and Dilley, 1976 for reviews). were established in newborn rats of trpptophan-deficient and normal mothers by immunofluorescence. 2. Materials and Methods The tryptophan-deficient diet was based on that used by van Sallnlanu et al. (1959) the calculated L-tryptophan content was reduced to @7Oq/,.The diet contained soya
but
* Present address: Department of Histology 2006, New South Wales, Aust’ralia. 0014-4835/79/050533+06 c
and Embryology,
$01.00/O
The University
of Sydney, Sydney
0 1979 Academic Press Inc. (London) Limited 533
A34
,J. 11’. MrAi’OY,
L. ,J. PALFREY
ASD
R. VAN
HEYNINGEK
bean protein 4 :,A, gelatin 4%. casein acid hytlrolysate S’$&, corn meal 32q,, corn oil 10”~. sucrose 35.2yb, salts 6’$/,. u,L-methionine 0.4%, u,L-lpsine 0.2~/0, inositol O.l’:;, cliolitle chloride O-l;/, and vitamins. Litter mates of Wistar strain albino rats were used. Ha,lf were weaned onto the deficient diet alone and the other half onto the deficient diet, with L-tryptophan (2 g/kg). Food and water were given ad lib. After 4-5 weeks on the diet the rats were killed and the eyes prepared for histology. In another experiment female rats were placed on the deficient diet, and after 2 weeks were allowed to breed. The females that conceived were maintained on the diet. The newborn rats were killed and the eyes prepared for histology.
Eyes were fixed in Carnoy’s (3: 1 ethanol : acetic acid) for 30 min and ettlbetldetl in paraffin. Serial sagittal sections were cut at 5 ~111.Xea,surements and volume calculations were made from three sections from the middle of the lens (the largest sections). These were made according to method II of Wolpow and Marimont (1967) which assumes that t’lle lens is an ohlate spheroid. Measurements xnd then volume calculabions were made for each of the three sections. The average of these was taken as the lens volume. The volumes of five experimental and three control lenses from rats 4-5 weeks on the trvptophall-deficient diet as well aq volumes of four experimental and four control ne~~~.hortt rat lenses were c,alculat,ed.
Anti-crystallin antibodies prepared as described previously (McAvo~, 1978) were used to localize x-, /3- and y-crystallins in sections of eyes by the indirect, 1n~nlu~lofluoreacelIt technique of Weller and Coons (1954). Whole eyes and in some cases lens segments were fixed in Carnoy’s at room temperature for 30 min, routinely prccessed aud embedded in paraffin. Antibodies were applied to 4 pm sect.ions followed by goat anti-rabbit, gamma globulin antibody conjugated with fluorescein isothiocyanate (FITC, Nordic Imn~unological Laboratories, The Netherlands). To determine the specificity of t’he fluorescence the following two controls were carried out. Serum from non-immunized rabbit* was substituted for specific anti-crystallin antisera. The other control was to apply FITC directly to the section Gthout prior incubation it1 serum. In both controls no fluorescence \txs obser\-ed.
3. Results and Discussion
Lens volumes of tryptophan-deficient and control rats were calculated front sagittal s&ions. The volumes of two age groups of rats were calculated; newborn rats whose mothers were fed on the trpptophan-deficient diet for 2 weeks before conception anal rats fed on t#he diet from 4-5 weeks after weaning. The results are shown in Tat& T. In both experime.nts the lenses from tryptophan-deficient rats arc sttlaller than lenses froltl control animals. These volume differences bet,ween tryI’tol)ll~~tl-tleficient and controls are in general agreement with lerls weight differences (L)isch~ et al.. 1959; van Heyningen, 1976; Ohrloff et al.. 1978; Runce et al., 197X). Tbs. in t,oth c,xperiments reduced tryptophan intake resulted in slower than normal lctls gron-th.
We previously reported that. in tryptophan-deficient deeper into the lens and are 2- 3 times more numerous
rats, nucIeatec1 cells extend than in controls. In addition
(‘HASGES
IN
THE
TRYPTOPHAN-UEFIC’IENT
LESS
35
TABLE I
Lens volumes of tryptophan-dejicient and control rats of two age groups (stdard deviations included) Rats
Lens volume cont~rols
Newborn 1-5 weeks post weaning
I~S~W4 1’.7& 1.0
(mm”)
Tryptophan-deficient
1.0*0.5 8.6&0.9
nuclei with abnormal morphologies are present (McAvoy and van Heyningen, 1976). In the study reported here the cells with the abnormal nuclei are shown to have a variety of shapes and in general are much thicker than normal fibre cells (Fig. 1). In both anterior and posterior lens cortex there is an abundance of cells with abnormally large diameters. Also in some cases there are what appear to be areas of fibre cell decomposition. The cells have variable diameters and do not form regular rows as do the fibre cells in normal lenses (Fig. 2). They are probably the anterior and posterior ends of the large cells with abnormal nuclei. These large cells might be swollen fibre cells or the result of the fusion and decomposition of &re cells. Indeed, swelling may precede fibre fusion and decomposition.
F’rc:. I. (a) The lrns bow of a rat fed on the tryptophan-deficient diet for 5 weeks postweaning. Haematosylin. phase contrast, Y 120. (b) This urca is indicated on (a) and shows detail of the fibrt ccllx with ;tl~nwmal morphologies (arrow). Havmatnxylin, phase contrast, ,: 480.
FIG. 2. Anterior lens cortex of tryptophan-deficient and control rats, 5 weeks postweaning. Haomatoxylin, phase contrast, > 130. (a) Control rat. The diameters of fibre cells do not, vary to the extent shown in (b) and (c). Fibre cells are regularly packed into rows. (b) Tryptophan-deficient rat. Tho diameters of the fibre cells are abnormally large and very variable in size. (c) Tryptophan-deficient rat. Thr amorphous area (arrow) may represent, a region of fibre cell decomposition.
These ahxlornlalities are probably related to, if not the direct cause of. the inner reflecting surface within the lens as seen when these tryptophan-deficient rat eyes are viewed in the slit lamp (see van Heyningen. 1976).
Distribution
of qstallins
in the tryptophnn-dejcielzt
mt Lens
It was found that in thenormal 4-5-week-old rat lenses the localization of’ crystallins by inmumofluorescence gave inconsistent results. The main observatiom were that
(‘HAXGES
IN
THE
TRYPTOPHAN-DEFICIENT
LENS
537
after sections were treated with crystallin specific antibodies and FTTC a variable number of the superficial lens fibres fluoresced and the deeper lens fibres did not fluoresce. This was the case when all three groups of antibodies were used. The absence of fluorescence after antibody treatment has also been observed in central fibres of mature chick lenses (Ikeda and Zwaan, 1967 ; Rrahnia and vau Dooreuuiaalen. 1971). For this reasou a meaningful comparison between tryptophan-tlcficiellt, ant1 control lenses front this age group could not be made. The localization of crystallins in newborn rat lenses gave consistent results. Sagittixl sectiom of tryptophau deficient lenses treated with anti K-MltihJdieS showed apple ~reea fluorescence in all lens cells [Fig. 3(a)].
ITsiug mti P-antibodies the green fluorescence was not tlet’ected in all lens ~11~;. /j-(‘rystallirl was detected only iu the cytoplasm of fibre cells and in cells at t’he icus equator that hat1 just begun to elongate [Fig. 3(b)]. I!sing anti y-antibodies green fluorescence was onlv found in the cytoplz,sm of fitne cells. In this respect the distribution of y-was similar to /3- but different in that it was first clet,ect,ed in cells that had considerably elongated [Fig. 3(c)]. The tlistributions of the three major groups of crystallins in the smaller lenses of tryptophan deficient rats are essentially similar to that previously described in lenses from normal new-born rats (Mcdvoy, 1978). Thus, in newborn animals at least, try@olhan deficiency does not alter t’he sequence of changes in crystallin synthesis tlurin~ the formation of fibre cells. ACKNOWLEDGMENTS
The support of this research was provided by grant no. 1 ROl EY 01548-01 awarded by the N.E.I. to Ruth van Heyningen.
5x3
J. W. McAVOY,
L. J. PALFREY
AND
K. VAN
HEYKINCEX
REFERESCES Brahms, S. K. and van Doorenmaalen, I$:‘. J. (1971). llllrnnnofluoresuence studies of chick lens FlSC and sc-crystallin antigens during lens morphogenexis and development. Ophthtrl. fi’~s. 2, 344-67. of Bunce, G. E. and Hess. J. L. (1976). Lent,icular opacities in young rats as a ~OINY~LIHII~A maternal diets lo\\. in tryptophan and/or Vit,amin E. J. AVufr. 106, 222-g. Brmce, G. E.. Hess, J. L. and Fillnow. G. M. (1978). Investigat,ion of low tryptophan intlucrtl c*at,aract in weanling rats. Ezp. Eye Res. 26, 399-405. Clayton. R. 31. (1974). Comparative aspects of lens proteins. In ‘I’he Eye, Vol. 5 (&Is Davson. H. and Gmham, L. T.). Pp. 399-494. London: Academic Press. Dische, Z., Elliott, J.. Pearson, E. and Merriam. (:. IX. (1959). Changes in proteins and protriu synthesis: In tryptophane deficiency and radiation cataracts of rats. dm. J. Ophthrtl. 47, 36X-79. Harding, J. ,I. and Dilley, K. J. (1976). Sitructural proteins of the mammalian lens: a revie\\ with emphasis on changes in development, ageing and cat’aract. E’zp. Eye Res. 22, l-73. Ikeda. A. and Zwaan, J. (1967). The changing celiular localization of n-crystallin in the lens ot the chicken embryo, studied by immunofluorescence. Ded. Biol. 15, 348-67. Xcdvoy, J. FT. and van Heyningen, R. (1976). Changes in the cells of the lens bow and epitheliunl of tryptophan-deficient rats. Colloq. d’Iruernl 60, 245-50. MrAvoy, J. IV. (1978). Cell division, cell elongation and distribution of s(-, ,8- and y-orystallins in the rat lens. J. Ernbryol. Exp. Morph. 44, 149-65. Ohrloff, C., Stoffel, C., Koch, H.-R., Wefers, U.. Boars. J. and Hockwin, 0. (1978). Experimental cataracts in rats due to tryptophan-free diet. dbrecht. r. Gmefes. A4rch. Ophthnhrrol. 205, 73-9. Totter, J. R. and Day, P. L. (1942). Cataract and other ocular changes resulting from t’ryptophanc deficiency. J. AVutr. 24, 159-66. van Heyningen. R. (1976). Experimental studies on cataract. J~csf. Ophthdrnol. 15, 685-97. von Sallmann. L., Reid, RI. E., Grimes, P. -4. and (‘ollins, E. 81. (1959). Trpptophan-deficirn(,>cataract in guinea pigs. arch. Oph.thaZnzol. 62, (352 72. \Veller, T. H. and Coons, A. H. (1954). Fluorescent antibody studies with agents of ~~icellrc :lntl herpes zosfer propagated in vitro. Proc. Sot. E.rp. Viol. Jletl. 86, 789-94. \Volpow, E. 11. and Marimont, R. B. (1967). Mathematical approach to measurements on embryonic chick lens. J. Morph. 121, 71-80.