|Molecular Vision 2002;
Received 21 May 2002 | Accepted 17 December 2002 | Published 19 December 2002
Quantification of chick lens αA- and δ-crystallins in experimentally induced ametropia
Sophia Zaidi, Michelle
Senchyna, Jacob G. Sivak
School of Optometry, University of Waterloo, Waterloo, Ontario, Canada
Correspondence to: Michelle Senchyna, PhD, School of Optometry, University of Waterloo, Waterloo, Ontario, N2L 3G1; Phone: (519) 888-4567, ext 6547; FAX: (519) 725-0784; email: firstname.lastname@example.org
Purpose: The role of the lens in experimentally induced ametropia is not known. A recent study of the chick lens demonstrated optical quality deterioration with the induction of refractive errors, without alteration in lens morphology, size or shape. A change in lens gradient of refractive index (which is dependent on α-, β-, and δ-crystallin concentration and arrangement), could underlie this observation. The purpose of this work was to quantify the concentrations of αA- and δ-crystallin in lenses from chick eyes with induced high myopia or hyperopia.
Methods: White Leghorn chicks were unilaterally fitted on the day of hatching either with translucent plastic goggles to induce form-deprivation myopia (n=21) or with +15 D defocus goggles to induce hyperopia (n=14). The ungoggled contralateral eyes were used as controls. The chicks were refracted twice, once on the day of hatching and again seven days later, using streak retinoscopy. On day 7 chicks were sacrificed, lenses decapsulated, and soluble proteins were isolated. Western blot assays were optimized and used to assess crystallin concentration.
Results: Analysis revealed no significant difference in αA- or δ-crystallin concentration in lenses from eyes induced with form-deprivation myopia and hyperopia as compared to their respective control eyes. Analysis of the difference in medians of δ-crystallin between the control and treated groups of the myopia and hyperopia experiments revealed significance (p=0.030).
Conclusions: This study suggests that with the induction of ametropia, the increased lens spherical aberration previously noted is not due to a change in the absolute concentration of lens αA- or δ-crystallin. However, results suggest that the myopic and hyperopic treatments had different effects on lens δ-crystallin concentration. Further investigation is necessary to expand the current knowledge of the role played by the lens in experimental ametropia.
Numerous studies have used chicks as a model for ametropia development [1-3]. From such studies, it has been demonstrated that when specific refractive errors are induced, changes to globe length and/or changes to choroidal thickness take place . Changes to the cornea such as steepening and flattening have been reported in induced ametropia, although the results have been inconsistent . The role of the lens is also unclear [6-8]. Many studies involving chicks have reported no change to the lens when comparing ametropic and control eyes [9-11]. In one of theses studies, Pickett-Seltner  reported no difference in total soluble protein concentration when lenses from myopic and non-myopic eyes were compared . The method employed however (Lowry Assay), was not capable of quantifying the concentration of individual proteins and therefore potential changes in the gradient of refractive index (GRIN) would not have been detected. Priolo , using the chick model, found that the optical quality of the lens deteriorates (increases spherical aberration) with the induction of ametropia, while there was no change in lens morphology, size, and shape . Given the unexpected change in chick lens optics with induction of ametropia, the overall role of the lens in experimental ametropia remains unclear. Deterioration of lens optical quality could have been the result of a change to the GRIN, which is formed by lens crystallins. Crystallins are the main component of the lens, occupying up to 90% of all total soluble proteins. Crystallins are essential for the lens to maintain transparency, a result of their refractive, enzymatic, and chaperone properties . The objective of this study was to help clarify the role of the lens refractive index gradient in ametropia. Quantitative assays based on electrophoresis and western Blotting were optimized and utilized to quantify lens αA- and δ-crystallins in hyperopic and form-deprived (myopic) eyes in comparison to contralateral control eyes.
Monoclonal sheep anti-δ-crystallin and anti-αA-crystallin antibodies were kindly provided by Drs. J. Piatigorsky (NIH) and J. Horwitz (Jules Stein Eye Institute), respectively. Horseradish peroxidase (HRP) conjugated donkey anti-sheep immunoglobulin G (IgG) was obtained from Sigma-Aldrich (Oakville, ON, Canada). All pre-cast PhastgelTM 10-15% SDS gradient gels, SDS buffer strips, well combs, and ECL-PlusTM kits were obtained from Amersham-Pharmacia Biotech (Baie d'Urfe, QC, Canada). All other reagents were obtained from either Sigma-Aldrich or Bio-Rad Laboratories (Mississauga, ON, Canada) and were of the highest quality available.
Chicks (Gallus gallus domesticus) were obtained on the day of hatching from a local hatchery (Big Four Chicks, New Hamburg, ON, Canada). They were housed in stainless steel brooders on a 14 h light/10 h dark cycle at a constant temperature of 32 °C. Chicks were supplied chicken feed and water ad libitum and were sacrificed after 1 week with CO2 asphyxiation.
Chicks were refracted on the day of hatching using a Welch AllynTM streak retinoscope. Retinoscopy was performed on the chicks using only the vertical streak in the horizontal meridian under the assumption that no astigmatism was present . Chicks were awake and unanaesthetised, and assumed to be focused on a point beyond the retinoscope.
The chicks (n = 21) were unilaterally fitted on the day of hatching with transluscent plastic goggles to induce form-deprivation myopia. A second set of chicks (n = 14) were goggled with +15 D defocus goggles to induce hyperopia. Goggles were cleaned three times per week with water and cotton tipped swabs. Goggles were removed after seven days and chicks were refracted again to determine the new refractive error.
Immediately following CO2 asphyxiation, eyes were removed, lenses were dissected, decapsulated and frozen at -70 °C. Soluble proteins were isolated by mechanical disruption of the lens followed by centrifugation [15,16]. Specifically, 200 μl of cold homogenization buffer (20 mM Tris-HCl, pH 7.5, 80 mM NaCl, and 1 mM EDTA) with 0.07% (v/v) β-mercaptoethanol was added to each lens, which was then homogenized using a Polytron PT10/35 homogenizer (Brinkmann, Westbury, NY), at a rheostat setting of 10 for 30 s followed by centrifugation in an EBA 12R centrifuge (Heittch, Tuttlingen, Germany) at 16,070x g for 30 min at 4 °C.
The concentration of total protein in each supernatant was determined by the Bradford Assay  using bovine serum albumin (BSA, Sigma-Aldrich, Oakville ON, Canada) as a standard and Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories, Mississauga, ON, Canada). Reactions were read on an Ultraspec 2000 spectrophotometer (Amersham Pharmacia, Uppsala, Sweden). Subsequently, lens samples were divided and vacuum centrifuged to dryness with a Savant Speed Vac (Halbrook, NY). Dehydrated samples were stored at -70 °C  for at least 24 h and to a maximum of 3 months.
Electrophoresis and immunoblotting
Frozen lens samples used for αA- and δ-crystalllin analysis were rehydrated with dilution buffer (50 mM Tris-HCl, 20 mM EDTA, 0.15 M NaCl, pH 7.4) to an initial concentration of 1 μg/μl total protein in all cases unless otherwise specified.
General procedure for SDS-PAGE and western blotting
All SDS-PAGE and semi-dry electrophoretic transfer to polyvinylidine difluoride (PVDF) membranes was performed using the PhastSystemTM (Amersham Pharmacia, Baie d'Urfe, QC, Canada). Each lens sample was further diluted 1:1 with loading buffer (50 mM Tris-HCl, pH 7.4, 10% [v/v] SDS, 1 mM EDTA, 0.02% [v/v] bromophenol blue, and 5% [v/v] β-mercaptoethanol), boiled for 3 min, applied to 8-well combs and loaded onto 10-15% SDS gradient gels (Amersham Pharmacia, Baie d'Urfe, QC, Canada). Proteins were transferred via semi-dry electrophoretic transfer to PVDF membranes (Bio-Rad Laboratories, Mississauga, ON, Canada) which were then blocked overnight in Tris-Buffered saline (TBS, 50 mM Tris, 100 mM NaCl, pH 7.4) containing 0.05% Tween-20 (TBS-T) and 10% skim milk powder (w/v). Antibody incubations (primary and secondary) were performed in TBS-T containing 5% (w/v) skim milk powder. Immediately following chemiluminescent detection with ECL-PlusTM (Amersham Pharmacia, Baie d'Urfe, QC, Canada), blots were imaged using a Storm 840TM Imaging System (Molecular Dynamics, Sunnyville, CA), and each band was quantified using ImageQuant 5.1TM software (Molecular Dynamics). Specifically, chemiluminescent signals were captured by placing a box of fixed dimensions around each of the eight bands on an image. Box size was chosen based on the largest band and was made to perfectly enclose the entire band. The resulting OD reading of each boxed region was used for quantitation of the signal. As OD readings were taken in arbitrary units, direct blot-to-blot comparisons were not valid. Hence data was compared using relative ratios as is described below.
Titration of lens proteins to determine linearity of αA- and δ-crystallin concentration with optical density
In order to determine the concentration of total protein required for quantitative western blot experiments, OD measurements of αA- and δ-crystallin bands obtained from increasing concentrations of total soluble protein were analysed. Protein concentrations between 0.001 and 0.16 μg/μl were subjected to SDS-PAGE and transferred to PVDF membranes. Blots were incubated with primary anti-αA-crystallin antibody for one h (1:1,500) or anti-δ-crystallin for two h (1:1,000). Subsequently, blots were incubated for one h with donkey anti-sheep-IgG-HRP antibody (1:6,000). Following chemiluminescent detection, optical densities were graphed against the amount of protein loaded, with the line of best fit inserted using linear regression analysis. From this range, a mid-point was chosen with the corresponding x-value representing the amount of total protein to load for all quantitative/comparative experiments.
Assessment of αA- and δ-crystallin concentration in lens extracts
Lens total soluble proteins at a concentration of 0.06 μg/μl or 0.05 μg/μl for αA- and δ-crystallin respectively, were subjected to SDS-PAGE and were transferred to PVDF membranes and incubated with appropriate antibodies as described above.
Two blots were run simultaneously, one with 7 treated lens samples from the goggled right eyes (OD) and the other with 7 control lens samples from ungoggled left eyes (OS). Lane 1 in each blot was reserved for the reference, an untreated lens sample with which all band densities were normalized. This procedure allowed for blot-to-blot comparisons of data collected using arbitrary OD readings. All experiments were performed in triplicate.
All statistical analyses were performed using SigmaStatTM 1.0 statistical software. Refractive errors of control and treated eyes were compared using a Student's t-test, where p<0.05 was significant. Western blotting data, which became non-parametric following normalization, were statistically analysed using the Wilcoxon Signed Rank Test, where p<0.05 was significant. Differences in medians between hyperopic and myopic experiments were analysed using the Mann-Whitney Rank Sum test, where p<0.05 was significant.
Chick refractive state
At Day 0, chicks were mildly hyperopic, with refractive errors in diopters (D) of +2.0±0.3 (SEM) for left eyes (OS) and +2.4±0.3 for right eyes (OD). Treated eyes (OD) of chicks (n=21) fitted with transluscent plastic goggles produced a final refractive error that was a significant myopic shift (-14.2±0.9 [SEM]) relative to the contralateral control eyes (OS, n=21) which were left ungoggled (+2.7±0.2 [SEM]). The eyes (OD) of chicks goggled with convex +15 D defocus goggles (n=14) produced higher final refractive errors (+8.5±0.5 [SEM]) than the ungoggled control eyes (OS, +3.5±0.3 [SEM]).
Assessment of αA-crystallin concentration in lens protein extracts from control and treated chick eyes
The αA-crystallin assay optimized in this study demonstrated linearity between optical density and protein concentrations between 0.01 to 0.16 μg (Figure 1). The αA-crystallin band was evident at 19 kDa  and non-specific binding of the antibody was evident with the presence of a less intense αB-crystallin band at 20 kDa (Figure 1) [18,19]. Based on this data, the optimum concentration of protein to load in quantitative western blot experiments was chosen to be 0.06 μg. Analysis of western blot data demonstrated that although the medians from the treated samples were slightly higher than the control in both cases, both myopic and hyperopic experiments showed no significant difference between the control and treated groups (Figure 2).
Assessment of δ-crystallin concentration in lens protein extracts from control and treated chick eyes
The quantifiable range of the δ-crystallin assay was determined to be between 0.001 to 0.15 μg of total soluble protein. A single band of immunoreactivity was observed at approximately 48 kDa [18,20], with streaking observed at higher concentrations. From this analysis, 0.05 μg of total protein was chosen as the concentration to load on subsequent experimental blots. The immunoblots comparing lens δ-crystallin in treated and control samples each showed one δ-crystallin band in each lane at approximately 48 kDa, with lane 1 being the reference which the samples were normalized to. An example of a representative western blot can be seen in Figure 3. The lenses from myopic and hyperopic eyes showed a higher and lower (respectively) δ-crystallin ratio compared to the control, although the differences were not significant (Figure 4). Comparison of relative median difference (treated compared to control) between the hyperopic and myopic study revealed significance, which suggests that the two treatments had opposite effects on the lens.
This is the first study comparing individual crystallin classes in chick lenses from eyes with induced refractive errors. As such, data comparisons with other studies cannot be made.
Although no significant difference in δ-crystallin concentration was observed between treated (hyperopic and myopic) groups and their respective controls, there was a difference between relative medians for myopic and hyperopic lenses. This finding seems to suggest that these two treatments had opposite effects on δ-crystallin concentration. Myopic treatment resulted in greater variation in the differences in ratios of δ-crystallin from control than the hyperopic treatment.
To further explore the role of crystallins in induced refractive errors, the procedures outlined in this study could be modified to quantitatively assess crystallins other than αA- and δ-crystallin. Although this study found no change in crystallins with induction of ametropia, it should be noted that αB- and β-crystallin concentrations were not assessed in this study. Additionally, post-translational modification of any of the crystallins could effect the refractive index distribution of the lens and have local ramifications that were not explored in this study. We have presented a novel approach to opening this area. Our findings at this time suggest that a factor other than αA- and δ-crystallin concentrations may be responsible for the increased spherical aberration of the lens. However, clearly, further work in this area is necessary to fully understand the role of the lens in ametropia.
We would like to thank Drs. J. Horwitz (Jules Stein Eye Institute) and J. Piatigorsky (National Eye Institute) for providing the anti-αA-crystallin and anti-δ-crystallin antibodies used in this study.
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