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Investigative Ophthalmology & Visual Science, Vol. 29, No. 2, February 1988
Copyright © Association for Research in Vision and Ophthalmology
An Ultrastructural Analysis of Plasma Membrane
in the U18666A Cataract
J. R. Kuszak,* A. R. Khan,* and R. J. Cenedellaf
Because the cholesterol concentration of lens fiber cell membrane in general and lens intercellular
junctions in particular is comparatively high, it is likely that it plays a major role in maintaining these
structures. In addition, the high concentration of cholesterol in fiber cell membrane is also likely to
influence membrane fluidity. Subcutaneous injections of U18666A (3 beta-(2-diethylaminoethoxy)
androst-5-en-17-one HC1) into rats effects: (1) a blockade of sterolgenesis in the lens; (2) a depletion of
lens fiber cell membrane cholesterol; and (3) the development of irreversible nuclear cataracts. In the
present study we have analyzed the ultrastructure of lens fiber cell membrane from U18666A cataracts
by the freeze-etch technique. Whereas it has been previously demonstrated that intercellular junctions
comprise approximately one-third of the intermediate cortical fiber cell membrane in adult rats, these
junctions were completely absent between comparable fiber cells taken from opaque regions of the
U18666A cataractous lenses. There was also a concomitant increase in the extracellular space between the opaque fiber cells and a substantial redistribution of intramembrane proteins in the exoplasmic and protoplasmic faces of these cells. These findings support a "hypothesis" that inhibition of
endogenous lens cholesterol production leads to damage and/or degeneration of lens fiber cell membrane in general and in intercellular junctions in particular, resulting in the production of an irreversible nuclear cataract. Invest Ophthalmol Vis Sci 29:261-267,1988
in sterol synthesis in the lens8; (2) a decrease in the
sterol to phospholipid ratio in the cortex9; (3) a retardation of lens growth; and (4) the development of
irreversible nuclear cataracts.10 The initial opacification of the lens nucleus in the U18666 A cataract is of
interest because cholesterol synthesis is nonexistent
in this the oldest region of the lens.4 Since lens intercellular junctions are believed to be important in
providing patent pathways for passive movement of
nutrients carried by the aqueous humor from the periphery of the lens to its center, and because the high
concentration of cholesterol within these junctions
suggests a role in their structural integrity, we questioned whether or not the ultrastructural integrity of
lens intercellular junctions was altered by treatment
with U18666A. In the present study we have employed the freeze-etch technique to analyze the ultrastructure of lens fiber cell membrane from opaque
and non-opaque regions of U18666A cataractous
lenses.
The plasma membrane of lens fiber cells is extremely rich in cholesterol. The molar cholesterol to
phospholipid ratio is as high as 3.5.' Alcala et al2 have
reported that the cholesterol content of lens intercellular junctions (lens gap junctions) is also very high.
Therefore, they suggested that cholesterol may play a
role in stabilizing the lens intercellular junctions.
Cholesterolgenesis in the lens epithelium and lens
cortex is believed to supply this sterol for the production of the enormous amount of lens fiber cell membrane formed by the lens throughout a lifetime34 because the lens, an avascular, stratified, epithelial
organ, is essentially isolated from circulating lipoproteins by the ocular humors.5
Treatment of rats with U18666A (3 beta-(2-diethylaminoethoxy) androst-5-en-17-one HC1), an inhibitor of 2,3-oxidosqualene cyclase and therefore cholesterol biosynthesis6'7 effects: (1) a marked decrease
From the *Department of Pathology, Rush-Presbyterian-St.
Lukes' Medical Center, Chicago, Illinois, and the fDepartment of
Biochemistry, Kirksville College of Osteopathic Medicine, Kirksville, Missouri.
Supported by NIH NEI grant nos. EY-06642 to JRK and
EY-02568 to RJC and the Otho Sprague Memorial Institute.
Submitted for publication: April 16, 1987; accepted July 14,
1987.
Reprint requests: Dr. J. R. Kuszak, Department of Pathology,
Rush-Presbyterian-St. Lukes' Medical Center, 1753 West Congress
Parkway, Chicago, IL 60612.
Materials and Methods
All experimental procedures reported here conformed to the ARVO Resolution on the Use of Animals in Research. Sprague-Dawley rat pups (Hilltop
Lab Animals, Springdale, PA) of either sex were injected subcutaneously every other day with 10 mg/kg
of U18666A (in olive oil) beginning at 1 day of age.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Februory 1988
Age-matched controls received no treatment. By 3
weeks of age, permanent nuclear cataracts developed
in 50-80% of treated rats.8
Freeze Fracture
The lenses examined by the freeze-etch technique
measured approximately 3 mm in the equatorial axis
and approximately 2.75 mm in the antero-posterior
axis. U18666A cataractous lenses had an opacity extending from the center of the lens to approximately
500 ixm from the lens surfaces at the equator and to
approximately 300 nm from the lens poles. Control
lenses were completely transparent before, during
and after fixation. The areas of lenses examined by
the freeze-etch technique consisted of approximately
1 mm3 pieces dissected from the middle segments of
intermediate cortical fiber cells, that is, greater than
500 nm from the lens surface in the equatorial plane
and at least 1000 ^m from the center of the lens in the
equatorial plane. Consequently, pieces from cataractous lenses were opaque while pieces from control
lenses were transparent. In addition, approximately 1
mm3 pieces of lens from the middle segments of superficial and developing fiber cells (within the first
500 /urn from the lens surfaces) of U18666A cataractous and control lenses were prepared for freeze-etch
analysis. In the cataractous lenses these pieces were
transparent though the remainder of the lens was
opaque.
Lenses were freeze-fractured as described previously. '' Briefly, the procedure was as follows: lenses
were fixed at room temperature in 2.5% glutaraldehyde prepared in 0.07 M sodium cacodylate (pH 7.2)
for 72 hr. Following an overnight buffer wash, lens
parts (1 mm3) were gradually infiltrated with glycerol
(25%) as a cryoprotectant. Lens parts were mounted
on standard Balzers (Hudson, NH) gold stubs and
frozen in liquid nitrogen-cooled Freon 23. Lens parts
were fractured and replicated at — 115°C in a Balzers
301 freeze-etch unit according to standard techniques.12 Replicas were retrieved onto formvar
coated 100 mesh copper grids and examined in
a JEOL 100CX transmission electron microscope (Peabody, MA) at 80kV. TEM stereomicrographic pairs were prepared by tilting the stage
± 12.5°.
Quantification of IMP Density and Distribution
The density and distribution of intramembrane
proteins (IMPs) in non-junctional membrane areas
from replicas of control and U18666A treated lenses
were quantified on a Calcomp 9100 Series Digitizer
(Scottsdale, AZ). Replica plots (areas of membrane
measuring approximately 2 nm2 at XI 00,000 magnification) were used for the quantification of the num-
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Vol. 29
ber of IMPs on the exoplasmic (EF) and protoplasmic
(PF) faces of control and U18666A treated lenses.
Results
A representative low magnification micrograph of
a freeze-etch replica made of control rat lens intermediate cortical fiber cell middle segments is shown
in Figure 1. The membrane exposed contained both
PF and EF areas. The characteristic narrowing of the
extracellular space at sites of lens intercellular junctions is apparent. The density and distribution of lens
intercellular junctions in this region of the control rat
lenses are consistent with previous reports that have
shown that approximately 32% of this membrane is
specialized as lens intercellular junction.1314
A representative low magnification micrograph of
a freeze-etch replica made of U18666A cataractous
lens intermediate cortical fiber cell middle segments
is shown in Figure 2. While the membrane exposed
contains both PF and EF areas of membrane the distinction between the two membrane surfaces as evidenced by their relative density of IMPs is markedly
less apparent than in control lens replicas. In all of the
membrane exposed from the opaque areas of
U18666A lenses we were unable to detect a single
lens intercellular junction. The fracture shelf between
the PF and the EF of these replicas was consistently
larger than in the control lens replicas, suggesting that
not only were the lens intercellular junctions completely eliminated but that the extracellular space was
concomitantly enlarged. In some replicas from the
U18666A cataractous lenses the extracellular space
was so large that it was not possible to reliably identify the PF and the EF (Fig. 3). These replicas were
not used for the quantification of IMP density and
distribution of IMPs on PFs and EFs.
The results of quantifying and comparing the density and distribution of IMPs in the PF and EF of
non-junctional membrane from replicas of control
and U18666A lenses is summarized in Figure 4.
These results clearly demonstrate that there is an increased density and a severely altered distribution of
IMPs in the PF and the EF of opaque fiber cells from
U18666A cataractous lenses.
A representative area of PF and EF membrane
from a transparent area of U18666A cataractous lens
is shown in Figure 5. While there are aggregates of
IMPs similar in appearance to lens intercellular junctions we do not consider these areas of membrane to
be junctional for the following reasons: (1) stereopair
micrographs of these aggregates revealed that the
shelf between the PF and EF was not uniform across
the extent of the plaque as is typical of an intercellular
junction1315; and (2) the particle packing of these ag-
No. 2
263
PLASMA MEMBRANE STRUCTURE OF U18666A CATARACT / Kuszok er ol.
D
D
D
O
D
O
o
o
o
D
O
O
0.5/im
Fig. 1. Transmission electron micrograph of a freeze-etch replica of the middle segment of control lens intermediate cortical fiber cell. Lens
intercellular junctions are identified by black stars (complete PF exposure), open black squares (PF and EF exposure) and open black circles
(complete EF exposure). X30,000.
gregates is considerably looser than is typical of lens
intercellular junctions.11131516 In addition, the density and distribution of IMPs in the PF and EF of
these cells was comparable neither to transparent
fiber cells from control lenses nor to opaque fiber
cells from U18666A cataractous lenses. The number
of IMPs on the PF of these cells was less than in
control lenses but greater than in U18666A treated
cataractous lenses. The number of IMPs on the EF of
these cells was greater than in control lenses but less
than in U18666A treated cataractous lenses.
Discussion
Freeze-etch analysis of the ultrastructure of membrane from opaque fiber cells of U18666A cataractous lenses has shown: (1) opaque fiber cells of
U18666A cataractous lenses are not conjoined by
any lens intercellular junctions; (2) opaquefibercells
of U18666A cataractous lenses are separated by en-
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larged extracellular spaces; and (3) opaque fiber cells
of U18666A cataractous lenses have a severely altered distribution of IMPs on their PFs and EFs. The
PF to EF ratio of IMPs in middle segments of intermediate cortical fiber cells U18666A cataractous
lenses is 0.72:1 whereas in control lenses it is 3.6:1.
Freeze-etch analysis of the ultrastructure of membrane from transparent fiber cells of.U18666A cataractous lenses demonstrates that: (1) lens intercellular
junctional morphology between these cells was altered with particle packing being looser and the narrowed extracellular "gap" being less uniform across a
junctional plaque; and (2) the density and distribution of IMPs between the PF and EF of these cells was
greater than in control lenses but less than between
opaque cells of U18666A cataractous lenses. Fiber
cells of U18666A cataractous lenses, essentially isolated from the cholesterol of circulating lipoproteins,
and incapable of producing endogenous cholesterol,
have plasma membrane with lower than normal
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / February 1988
Vol. 29
Fig. 2. Transmission electron micrograph of a freeze-etch replica of an opaque middle segment of U18666A treated lens intermediate
cortical fiber cell. Irregular extracellular spacing and a complete absence of lens intercellular junctions is apparent between the EF and PF.
X75.000.
amounts of cholesterol.9 This finding, along with the
results presented here, leads us to "hypothesize" that
the depleted cholesterol content of these fiber cell
membranes resulted in unstable intercellular junctions between these cells causing their eventual disassembly as well as a redistribution of the IMPs in a
more fluid membrane. However, since freeze-etch
methodology is a static technique, interpretation of
results as time-related phenomena is "purely speculative." But the fact that freeze-etch analysis of membrane from non-opaque fiber cells of U18666A cataractous lenses demonstrated possible partial disassembly of lens intercellular junctions and perhaps an
early stage of the redistribution of the IMPs within
the PF and EF is consistent with our interpretations.
It is interesting to note at this point that aging studies
have shown that both a demise in the number of
functional gap junctions and a redistribution of IMPs
within EF and PF, comparable to the results shown
here, are characteristic of aging cells.17'18
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Furthermore, the results presented here are consistent with the results of ongoing work (Rintoul and
Cenedella, unpublished observations) that show that
a reduction of approximately one-third of the cholesterol concentration in lenses following U18666A
treatment9 effects a change in membrane fluidity.
Fluorescence depolarization of trans parinaric acid in
corticalfibercell plasma membrane is approximately
20% higher and 20% more ordered than in control
membrane. Also, there is no apparent difference in
the anisotropy of the probe between protein-free liposomes prepared from control and cataractous lens
membrane. We interpret these preliminary findings
to indicate that protein orders this probe in lens fiber
cell membranes and that the protein of cataractous
lens fiber cell membrane is more accessible to the
probe (thus the higher order). The altered membrane
fluidity might also explain the observation that extensive proteolysis of MP26 to MP23-24 is also seen
in lenses treated with U18666A.19'20 A membrane
No. 2
PLASMA MEMBRANE STRUCTURE OF U18666A CATARACT / KUSZQK er ol.
265
Fig. 3. Transmission electron micrograph of a freeze-etch replica of an opaque middle segment of U18666A treated lens intermediate
cortical fiber cell. The extracellular space (rows of black stars) is so enlarged that identification of EF and PF is less than certain. X75,OOO.
bound protease could have greater access to MP26 in
a more fluid membrane.18
A causal relationship between the development of
a nuclear cataract in U18666A treated lenses and the
absence of lens intercellular junctions between nuclear fiber cells of these lenses might be explained in
one of two ways.
First, lens intercellular junctions are generally accepted to be one of two pathways in the lens for intercellular distribution of nutrients carried in the
aqueous humor."'13"15'21"25 The other pathway for
intercellular communication in lenses is the recently
described cell-to-cell fusion zone, a large patent pathway formed by the fusion of two neighboring fiber
cells.15 If cholesterol has a role in maintaining the
integrity of lens intercellular junctions then a loss of
cholesterol would reduce the passive transfer of nutrients to the opaque nuclear fiber cells of U18666A
cataracts formerly conjoined by lens intercellular
junctions. While cell-to-cell fusion between nuclear
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lens fiber cells of these cataractous lenses would presumably still maintain a degree of intercellular communication in lenses that have lost their intercellular
1,200-i
1.014
- i
a.
3
U18BB6A TREATED
IMPs war* counted in a total ol 56.03 and 19.i4p 2 of non-iunctlonal control
fn = 44 replica plotal and U18666A traated In.23 tapllca plotal membrane,
respectively.
Fig. 4. Histogram demonstrating the distribution of IMPs in
non-junctional EF and PF membrane from the middle segments of
control and U18666A treated lens intermediate cortical fiber cells.
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INVESTIGATIVE OPHTHALMOLOGY G VI5UAL SCIENCE / February 1988
Vol. 29
Fig. 5. Transmission electron micrograph of a freeze-etch replica of a transparent middle segment of U18666A treated lens superficial
cortical fiber cell. Aggregates of IMPs in the PF that appear to be junctional (black on white rectangle) have a much looser particle packing
arrangement. X75.000.
junctions, it should be noted that lens intercellular
junctions may serve as the initiation sites for cell-tocell fusion in the lens.15 It is not known if cell-to-cell
fusion zones in lenses are permanent. However, cholesterol has been ascribed a major role in directing
and maintaining specialized sites of membrane for
fusion in other tissues.26"29 Therefore, a reduction in
lens cholesterol might also lead to a reduction in the
integrity of cell-to-cell fusion zones in the lens. It has
been proposed that a lack of nutrition to fiber cells
anywhere within the lens would result in the loss of
lens transparency.24'30'31
Second, a lack of lens intercellular junctions between nuclear fiber cells and the presence of lens intercellular junctions between cortical lens fiber cells
would result in a loss of the uniform packing arrangement of lens fiber cells thought to be necessary for the
lens to be transparent. 32 " 34 As rays of light pass
through the lens they encounter in order membrane,
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cytoplasm, membrane and extracellular space, each
having a different refractive index. It has been proposed that by essentially eliminating the extracellular
space between the closely apposed junctional membrane, the membrane becomes doubled in thickness
resulting in the system (the lens) being effectively
transformed into a series of coaxial refractive surfaces, thereby producing transparency. Thus, any
substantial increase in the extracellular space between
fiber cells would increase diffraction, thereby reducing transparency. If the cholesterol content of fiber
cell membrane is essential to the integrity of lens fiber
cell membrane in general and lens intercellular junctions in particular then loss of cholesterol could presumably alter the highly regular spacing of fiber cells
by causing disassembly of the lens intercellular junctions.
Key words: lens, plasma membrane, intercellular junctions,
gap junctions, cholesterol, cataracts
No. 2
PLASMA MEMBRANE STRUCTURE OF U18666A CATARACT / Kuszak er ol.
Acknowledgments
The authors wish to acknowledge the excellent technical
assistance of Ms. Candace Ennesser and Mrs. Kathleen
Chapman.
18.
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