Denucleation process of the lens*
Toichiro Kuwabara and Masatoshi Imaizumi
Denucleating process of the mouse lens has been studied electron microscopically. The -first
step appears as a transformation in which the appearance of the nucleus becomes more like
that of the cytoplasm. When the appearance of both nuclear and cytoplasmic substances become indistinguishable, the nuclear membrane disappears without disturbing the size or other
structure of the cell. Extrusion of the nuclear substance or degeneration of the nucleus are not
observed during the denucleation. Fragments of nucleolus remain within the denucleated lens
for a long period of time. Disturbance of the denucleation process may cause certain cataractous
changes of the lens.
Key words: nucleus, nucleolus, denucleation, lens, development of lens,
maturation of lens cell, mouse.
A
One of the most unique characteristics
in these cells is that they are anuclear in
the mature form. Although the denucleation process of the erythrocyte,2~5 and the
details of the developmental process of the
lens,6'10 have been described, the denucleation mechanism of the lens cell has not
been studied. This process is understood
simply as degeneration. The present fine
structural study in the mouse lens has revealed that the denucleation appears as a
chemical transformation of the nuclear substance into the cytoplasm rather than a
degenerative change.
number of morphologic and metabolic similarities between lens cells and
erythrocytes have been pointed out. These
similarities between the two cells, particularly in the biochemical aspects, have been
discussed extensively and have been a
basis of a symposium on the red cell and
lens.1
From-the Laboratory of Vision Research, National
Eye Institute, National Institutes of Health,
United States Department of Health, Education, and Welfare, Bethesda, Md.; and Howe
Laboratory of Ophthalmology, Harvard Medical
School, Boston, Mass.
Part of this work was supported by a United
States Public Health Service Research Grant
EY00425, from the National Eye Institute
(Howe Laboratory).
Submitted for publication May 7, 1974.
Reprint requests: Dr. T. Kuwabara, National Eye
Institute, Building 6, Room 213, National Institutes of Health, Bethesda, Md. 20014.
°The summary of this study was presented at the
annual meeting of the Association for Research
in Vision and Ophthalomolgy, Sarasota, Fla.,
April 24, 1972.
Materials and methods
The developing albino mice (Charles River)
were used in this study. Also, several lenses of the
adult human and monkey were studied similarly
for comparison with the developing mouse lens.
Sixteen- and 18-day-old embryos were obtained
by killing three each of pregnant mice on the
corresponding gestation date. Postnatally developing mice were obtained from two different litters
on the first, third, seventh, and fourteenth days.
Animals were killed by sodium pentobarbital injections. The enucleated eyes were fixed in 4 per
973
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Investigative Ophthalmology
December 1974
974 Kuwabara and Imaizumi
Results
Fig. 1. Equatorial zone of an 18-day-old embryo
mouse lens. The elongating cells possess nuclei.
Nuclei in the deeper zone are in a round shape.
One micron epon section, toluidine blue stain.
x500.
cent glutaraldehyde solution in 0.15 M phosphate
buffer (pH 7.2) at room temperature. After about 10
minutes fixation, the cornea and the posterior portion of the eye were discarded. The lens and attached ciliary epithelium were fixed for 24 hours in
the same solution. The well-fixed lens was cut into
quadrants and then trimmed into smaller pieces.
The attached ciliary epithelium and hyaloid vessels were found to be useful landmarks in keeping
the orientation of the tissue. Pieces not larger than
0.2 mm.s were carefully obtained from the bow
area and were postfixed in 1 per cent osmium
tetroxide in the same phosphate buffer solution
for one hour at 4° C. The tissue was dehydrated
in a series of graded ethyl alcohol, treated with
propylene oxide, and then was embedded in an
epoxy resin, following Luft's method.11 The block
was re-oriented to show the longitudinal view of
the lens cells at the bow zone. Thick sections were
stained with toluidine blue and were examined
light microscopically. Thin sections were stained
with uranyl acetate and lead citrate and were
examined by a JEM 7 electron microscope.
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The earliest lens is a cyst of a single cell
layer which is developed by invagination
in and pinching off from the ectodermal
layer. The cyst space is filled rapidly with
elongating posterior cells and the lenticular
tissue is formed.
All the lens cells are nucleated until the
embryonal sixteenth day. However, soon
thereafter, elongating cells begin to lose
their nuclei and they separate from the
basal attachment at the posterior central
portion. On the eighteenth day, mature
lens fibers are found to be abundant in the
enlarged central portion, but still a great
number of nucleated cells are found in the
equatorial zone (Fig. 1). On the first postnatal day, the lenticular nucleus and the
bow configuration become apparent. By
the postnatal fourteenth day, the maturation of the lens appears to be completed.
No further alteration of the cytologic appearance occurs.
The denucleation process first occurs in
many cells in the central zone of the 16- to
18-day-old embryonal lenses and localized
at the inner bow zone after birth. Although
the number of denucleating cells decreases
drastically within these two days, an identical cytologic process is found in the later
developing stages. Since different stages of
the denucleation process are seen in all
lenses, the findings are described according to the location rather than to the age
of the animal.
The nucleus of the anterior lens cell
(epithelium) is round in shape and consists of coarse but diffuse chromatin
granules. With the elongation of the lens
cell, the granular crystalline substance,
which accumulates in the cytoplasm beginning at the earliest stage of development, becomes denser and the microorganelles begin to disappear. The cells at
the bow zone have uniformly granular
cytoplasm containing a small number of
mitochondria, rough endoplasmic reticulum, Golgi apparatus, and free ribosomes.
The elongated nucleus is made of coarse
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Denucleation process of lens 975
Fig. 2. Bow zone of a newborn mouse lens. The lens cell contains fine granular substance and
ribosomes. Micro-organelles are sparse. Nuclei are oblong in shape and contain course chromatin granules. Nucleolus is prominent. x9,800.
chromatin granules which aggregate into
small groups in several locations (Fig. 2).
Many large nucleoli measuring about lju.
in diameter are found regularly. The nucleolus consists of aggregations of granular
RNA particles (each particle measures
about 150 A in diameter) and fibrillar
background component (Fig. 3). The cells
in the slightly deeper bow zone contain
denser granular cytoplasm and less microorganelles. The nucleus is still elongated
but its chromatin substance becomes finer
and more homogenous. Similarly, the nucleoli begin to lose their original structure
and the RNA granules become fine and
denser (Fig. 4). Many equatorial lens cells
of the 18-day embryo and cells in the
superficial cortex of the adult lens show
these changes.
Cells in the deeper zone show homo-
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geneously fine granular cytoplasm which
has slightly increased electron density but
has lost most of the •micro-organelles (Fig.
5). These cells maintain the basal attachment with the capsule near the pole of
the posterior surface and extend their
apical ends directly inside the anterior lens
cells at the anterior portion. The nuclei of
these cells become round again, although
the cell bodies are extremely long. The
fine and uniform nuclear substance becomes almost indistinguishable from the
surrounding cytoplasm in appearance
(Fig. 6). The nucleoli appear to fuse into
larger masses. Frequently, the extremely
dense nucleolar substance surrounding the
clear zone, from which fibrillar structure
has disappeared, shows a spoke-like configuration (Figs. 4 through 6). These find^
ings are seen most frequently during the
976 Kuwabara and Imaizumi
Investigative Ophthalmology
December 1974
Fig. 3. A nucleolus in the nucleus of a bow cell
of a newborn mouse. The nucleolus consists of
RNA particles (arrows), homogeneous fine granules, and electron-lucent fibrillar background.
x21,600.
Fig. 4. A nucleolus of the deep bow cell of a newborn mouse lens. The nucleolar granules are fine
and denser. They are forming a wheel configuration. x31,300.
first few postnatal days and in cells in the
deeper bow zone of the adult lens.
The next step of the denucleation appears to be a removal of the nuclear envelope. The nuclear membrane fragments
and converts into vesicles at the time when
the nuclear and cytoplasmic substances
become identical in appearance (Fig. 7).
Although the original site of the nuclear
envelope is recognizable by chains of
vesicles which remain for a short while,
the nucleus itself is no longer distinguishable from the surrounding cytoplasm
either by electron microscope or by histologic staining, including Feulgen reaction. Eventually the vesicles disappear
from the cytoplasm completely without any
sign of degeneration or extrusion of the
nuclear substance. The total volume of the
cell appears to remain unchanged during
this process. Also, no excessive extracellular substance is found in this stage. The
electron-dense nucleolus substance is fragmented into thread-like bodies which remain within the denucleated cell body,
first in the original nuclear location, and
then are scattered in the homogenous cytoplasm in the later stage (Fig. 8). The
scattered fragments of the nucleolar substance are of various sizes and are found
occasionally in the mature lens cells, Large
nucleolar substance is visible in paraffin
sections as hematoxylin-staining positive,
but Feulgen-reaction negative, particles.
No degeneration, karyolysis, karyorrhexis,
or pyknosis is observed in these lens cells.
In the deep bow cells occasionally there
is coarse nuclear substance, presumably
RNA particles, which aggregate around
the pore of the nuclear envelope of the
rounded nucleus (Fig. 9). The picture
suggests that the nuclear substance is
streaming out through the pore. However,
no shrunken nucleus or collapsed nuclear
membrane is found in the vicinity. Also,
in the stage immediately prior to the removal of the nuclear membrane, large RNA
particles appear to reform within the
densely fine nucleolar substance (Fig. 8).
The particles seem to be scattered into
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Denucleation process of lens 977
Fig. 5. Deep bow zone of a three-day-old mouse lens. The lens cell consist of fine granular
substance. Almost no micro-organelles are found in the cytoplasm. The nuclear substance is
also dense and fine. Nucleoli are extremely dense and forming wheel configurations. x8,200.
the cytoplasm by completion of the denucleation.
After the denucleation, the cytoplasm
becomes completely homogenous with a
fine granular crystalline substance. Except
for a few ribosome particles, microorganelles are not seen in these cells. The
denucleated cells appear to be removed
from the basal attachment at the posterior
capsule and become the mature lens fiber.
The apical ends also appear to be pushed
into the deeper zone.
Discussion
The unique sequence of the denucleation process of the lens cell uncovered in
the present study is summarized schematically in Fig. 10. The first step appears as a
transformation in which the appearance of
the nucleus becomes more like that of the
cytoplasm. In addition, there is the removal of the micro-organelles. When the
appearances of both nuclear and cytoplasmic substance become indistinguish-
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Fig. 6. The nucleus of the lens cells in the deepest
bow zone of a three-day-old mouse. The nucleus
is round in this longitudinal section of the cell.
The nuclear and cytoplasmic substances are almost indistinguishable. The nuclear envelope is
slightly dilated. The nucleolus is large and
prominent, xl0,000.
978 Kuwabara and Imaizumi
~i
Investigative Ophthalmology
December 1974
Fig. 7. Lens cells in the deep bow zone of a three-day-old mouse lens. The cytoplasm is not
distinguishable from the nuclear substance. The nuclear envelope is recognizable as a chain
of vesicles (arrows). Fragmented nudeoli are seen in the disappearing nucleus. x21}000.
able, the nuclear membrane disappears
without disturbing the size or other structures of the cell.
Since the chemistry of the crystalline
substance of the cytoplasm and the nucleic protein are not related, it is difficult
to figure out the detailed steps of the
chemical pathway by which the nuclear
material is incorporated into the cytoplasm. However, the morphologic appearance suggests- the possibility of this
transformation in the lens cell. Vesiculation and disappearance of the nuclear
membrane is a common finding during
mitosis. A similar process may be occurring in the denucleating lens cell.
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Hanna and O'Brien1- reported that the
lens cell nucleus is extruded during the
course of production of lens fibers. The
extrusion mechanism of the red cell nucleus has been demonstrated electron microscopically.2"^ The present observation
has failed to demonstrate extrusion of the
nucleus in the lens cell. Since the lens is
capsulated and encased within the eye
ball, extrusion may not be a suitable process for discarding the nuclear protein.
Morphologic changes of the nucleoli before the denucleation are striking. However, these changes of the nucleolus may
not be specific to the denucleating lens
cell. Similar alteration of the nucleolar ap-
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Denucleation process of lens
979
Fig. 8. Deep bow zone of a seven-day-old mouse lens. Fragments of nucleolar substance
are found in the cytoplasm of anuclear lens cells, RNA particles are seen in the vicinity of the
fragment (asterisk and insert), xl7,000; insert, x44,000.
pearance has been described in other
mammalian cells in normal and various
pathologic conditions.111'1 However, it is
interesting to find that the fragments of
nucleoli remain within the denucleated
lens cells for a long period of time. Although the fate of the fragmented nucleoli is not followed thoroughly in this
investigation, the fragments may remain
in the lens cell for an extremely long time.
The dense particles which are found commonly in the deep cortical cells of the
adult lens presumably correspond to these
fragments. Although degeneration is considered generally to be the main course
of the denucleation mechanism of the lens
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cells, no nuclear degeneration was observed in this study. The pyknotic nuclei
which are seen often by light microscopy
appear to be the large nucleolar particle.
The degenerating nuclear particles which
have been described by Kobayashi17 appear to be these nucleolar particles.
It is also interesting to find clusters of
ribosomes in the vicinity of the fragmented
nucleoli and around the pores of the nuclear membrane before the denucleation.
Similar findings have been described in
various cells.1*"-" Ribosome particles which
are seen in the anuclear lens fibers may
have come from these sources.
Although the present observation is
980 Ruwabara and Imaizumi
Investigative Ophthalmology
December 1974
Nu
0.5
Fig. 9. Deep bow zone of a three-day-old mouse. Clusters of RNA particles are seen around
the nuclear pore. The nuclear substance is not distinguishable from the cytoplasm. x78,000.
Fig. 10. Schematic drawing of the denucleating process. A, elongated nucleus in the superficial
bow cell. The nucleolus consists of RNA particles. B, slightly deeper zone of the bow area.
The nucleolus becomes denser. C, the nucleus becomes round in the deep bow zone. The
nuclear and cytoplasmic substances are not distinguishable. The nucleolus is markedly dense
and forms spoke-like configurations. D, the nuclear envelope breaks up into vesicles and the
nucleus disappears. E, fragmented nucleolar substances are found in the lens cells for a long
period of time.
made mainly in the developing mouse lens,
similar findings are regularly found in the
human and monkey lenses. A much slower
but identical process seems to be occurring
in the course of lens cell maturation in all
stages. Disturbance of this process may
cause pathologic conditions of the lens.
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It is noteworthy to see retention of nuclei
in certain cataractous conditions, such as in
rubella in the human21 and congenital
animal cataracts.-This study is dedicated to Dr. George K.
Smelser whose friendship and inspiration encour-
Denucleation 'process of lens 981
Volume 13
Number 12
aged us greatly. Dr. David G. Cogan's continuous
interest in this research is appreciated.
13.
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