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J. Embryol. exp. Morph. 75, 271-291 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
271
Retinal pigment epithelium: pattern of proliferative
activity and its regulation by intraocular pressure in
postnatal rats
By O. G. STROEVA 1 AND I. G. PANOVA
From the N. K. Koltzov Institute of Developmental Biology, USSR Academy
of Sciences, Moscow
SUMMARY
The postnatal proliferative activity of retinal pigment epithelium (RPE) cells and its dependence on intraocular pressure were studied using pHjthymidine and [14C]thymidine
autoradiography in normal and experimentally induced microphthalmic pigmented rats. The
regulation of RPE growth by intraocular pressure was shown to involve the control of the
number of binucleated cells by means of stimulation of cell entry into the S phase of the cell
cycle. Binucleated cells in the rat RPE are formed by acy to kinetic mitoses between days 2 and
9. The significance of the postnatal proliferation and formation of binucleated cells in the RPE
is discussed in terms of the specificity of the G2 phase for melanotropic hormone action on
RPE differentiation.
INTRODUCTION
Until recently it was believed that retinal pigment epithelium (RPE) cell
replication in rats is completed during intrauterine life (Puzzolo & Simone,
1979). We found that in newborn rats RPE cell proliferation resumes with a spike
on the third day after birth, and a considerable proportion of RPE cells become
binucleated during the first two postnatal weeks (Stroeva & Nikiphorovskaya,
1970; Marshak & Stroeva, 1973, 1974; Stroeva & Panova, 1976, 1980). By the
time of eye opening the proportion of binucleated cells in the RPE central zone
reaches 80 %, and then declines to 70 % in 1-year-old rats (Ts'o & Friedman,
1967; Stroeva & Brodsky, 1968; Stroeva & Nikiphorovskaya, 1970).
This decline in number of binucleated cells is accompanied by tri- and
tetranucleated cell formation (Fig. 1). Observation on adult mutant MSUBL rats
(Stroeva & Lipgart, 1968) showed that in the RPE of microphthalmic eyes the
proportion of multinucleated cells is less than that in normally sized eyes
(Stroeva & Nikiphorovskaya, 1970; Marshak & Stroeva, 1974) which led us to
suggest that proliferation and the occurrence of polyploidy in RPE cells are
1
Author's address: N. K. Koltzov Institute of Developmental Biology, Academy of
Sciences of the USSR, 26 Vavilov Street, 117808 Moscow (B-334), USSR.
272
O. G. STROEVA AND I. G. PANOVA
100'
1o5 0 ^
a
o
1
3
5
7 9 11 13 15
age in days
1
3
5 7 9 11 13
age in months
Fig. 1. The proportion of uninucleated (filled circles), binucleated (open circles),
trinucleated (filled squares), and tetranucleated (open squares) cells in the central
zone of the RPE throughout the postnatal life of rats.
Each point derived from cell counts on tangential sections of the RPE using ten
eyes, 1000 cells per eye (averaged data from Stroeva & Nikiphorovskaya, 1970;
Marshak & Stroeva, 1973; Stroeva & Panova, 1976).
controlled by intraocular pressure. The latter was shown to be a general factor
controlling eye growth in the chick embryo (Coulombre, 1956). In order to test
this suggestion we studied (1) the pattern of eye growth throughout life in rats;
(2) the characteristics of the cycling RPE cells during thefirst2 weeks after birth
(the period found to be critical for postnatal RPE differentiation), and (3) the
origin of binucleated cells. Microphthalmic eyes were induced experimentally
and the eye growth retardation effect on the RPE was then studied. This paper
presents the results obtained. Some of our data were briefly reported earlier
(Marshak, 1974; Panova & Stroeva, 1978; Stroeva & Panova, 1976,1980). The
present research intends to serve as a basis for studies with more analytical
methods.
Retinal pigment epithelium in postnatal rats
273
MATERIALS AND METHODS
Animals
Grey rats (Rattus norvegicus) with pigmented eyes randomly bred from the
colony of the Institute of Developmental Biology were used in the experiments.
Eye growth
Rats at postnatal ages ranging from 1 day to 1 year were killed by decapitation.
Following enucleation and fixation in neutral formalin+96 °ethanol+glacial
acetic acid (3:1:0-3) the eyes were washed in tap water and 70° ethanol, dried
on filter paper and weighed using analytical scales. Their scleral part was then
cut off under the ora serrata and also weighed. For every measurement point
between 10 and 45 eyes were used. Then the scleral parts of 3 to 7 eyes per agepoint were processed for histological treatment. In another experiment the
scleral part of freshly enucleated eyes from 1- to 15-day-old rats cut off under the
ora serrata, was incised at several places along the margin and laid flat on filter
paper. The flat preparations were immersed in the fixative mixture, washed and
photographed. The area of the scleral part of each eye was measured on the
photo by planimetry (10 eyes for each measurement point).
The gain in weight (AW/At) and in area (AS/At) of the scleral part between
different ages were calculated from the equations:
AW/At = W(n) - W(n -l)/t(n) - t(n -1),
(Eq. 1)
AS/At = S(n) - S(n - l)/t(n) - t(n - 7),
(Eq. 2)
and
where W(n) and S(n) are a weight and an area of the scleral part of n-day-old rat
respectively, and t(n) is the age of animals in days.
Microphthalmic eyes were obtained by surgical extirpation of the lens after
Coulombre & Coulombre (1964), who demonstrated the role of the lens in eye
growth. Rats were subjected to surgical manipulations at the onset of the second,
and at the end of the fourth postnatal days, under ether narcotization. Once the
skin above the left eye and the conjunctival sac wall had been cut with a pair of
scissors the lens was extirpated from the eye through a corneal linear incision,
by use of a glass needle. Thereupon the skin was sutured and the animals were
returned to the mother. The right intact eye of each animal was used as the
control.
An increase in area of individual RPE cells in normal and microphthalmic eyes
was described by the nuclear concentration in the central RPE zone. A square
ocular grid was projected on the slides under the light microscope at a magnification of 1000 x , and the number of nuclei per square was averaged by counting
fifty such fields per eye. Nuclear concentration was calculated from the equation
274
O. G. STROEVA AND I. G. PANOVA
of Abercrombie (1946). In all experiments a minimum of three animals was used
for each measurement point.
A utoradiography
For continuous labelling intact and operated rats were injected subcutaneously with [3H]thymidine (specif, activ. 9-1 Ci/mM. 1 /iCi/g) every 6 h for 19 h, and
decapitated l h after the last injection. The experiments were started at
10.00a.m. For histological treatment, the eyes were processed as described
above, then their scleral parts were dehydrated in serially graded ethanol and
chloroform and embedded in paraffin. Serial sections, 5^m thick, were cut
tangentially to the RPE in three neighbouring regions of the central (posterior)
zone of the globe and from the four quadrants of the sublimbal zone referred to
as peripheral. The zone between the central and peripheral ones was called
equatorial (Fig. 2). The general state of microphthalmic eyes was examined from
serial cross sections prepared in a plane parallel to the optic axis. Melanin was
decoloured on dewaxed sections with potassium permanganate, whitened with
oxalic acid and washed with tap water. Autoradiograms were prepared with a
liquid emulsion (type 'M', Nil Chimphoto, Moscow), and exposed for a month
at 4°C. The slides were developed in D19 developer (Schillaber, 1944) and
stained over the emulsion with Carrachi haematoxylin.
Proliferative activity
The proportion of uninucleated and binucleated cells as well as of labelled
nuclei was calculated from counts of at least 1000 cells per zone of each eye. Cell
Fig. 2. Schematic drawing of the scleral part of the eye. Open circles indicate the
zones from which tangential sections were cut for cytological study of the RPE cell
population: (1) peripheral zone; (2) equatorial zone, and (3) central zone.
Retinal pigment epithelium in postnatal rats
275
cycle parameters were derived graphically (Quastler & Shermann, 1959). Fourday-old rats received a single injection of [3H]thymidine, and then the fraction
of labelled mitoses (FLM) was plotted as a function of time. Mitotic figures from
middle prophase to telophase were scored (30-60 mitoses per eye) on tangential
sections from the central RPE zone. Unless specified all nuclei with five or more
silver grains over them were considered as labelled. In order to determine the
duration of mitosis (IM), 4-day-old rats were injected subcutaneously with 5 jug/g
of colchicine (Merk, BRD) and killed 3h later. A preliminary experiment
revealed the complete blockage of metaphases in the RPE at that time. The eyes
of three intact animals from the same litter provided material for the determination of the mitotic index (m), which was derived from total counts of 16 644 cells.
The duration of mitosis was determined from the equation tm = (m/mc)t, (Eq. 3),
where t is the effective time of colchicine action.
In order to demonstrate the mitotic origin of binucleated cells, the grain
density over the interphase nuclei of uni- and binucleated cells at 2 h and 27 h on
the FLM curve, as well as over each of the two nuclei in 100 binucleated cells at
27 h, was obtained. The grain density was derived as the number of silver grains
per nuclear area (calculated as that of an ellipse).
Double labelling with [3H]thymidine and [14C]thymidine was used in order to
determine the number of cell cycles between day 3 and day 9 after birth. Twentyone 3-day-old rats were each injected with a single dose of [3H]thymidine (specif,
activ. 12Ci/mM; ljuCi/g). Three rats were killed 2h after the injection. The
other animals were sacrificed 24, 48, 72, 96,120, and 144 h after [3H]thymidine
injection (three animals per day); 2h before decapitation they were each given
a single injection of [14C]thymidine (specif, activ. 54mCi/mM; 1/iCi/g). The
eyes of all rats were enucleated, fixed and processed for histological treatment
and autoradiography. Indices of labelled nuclei of uninucleated and binucleated
cells were calculated from counting at least 1000 cells per eye on tangential
sections at the central zone.
All quantitative data were processed statistically.
RESULTS
Eye growth
According to the weight data, the rat eye grows throughout the life of animals,
whereas the growth of its scleral part has a discontinuous character (Fig. 3). The
most intensive growth of the scleral part occurs from day 2 to day 5 after birth,
with a maximum on day 4 (Fig. 3B). By day 5 the weight of the scleral part
reaches half of that of the 1-year-old rat. The increase in weight of the scleral part
was also noted between the first to third and the seventh to twelfth months of age
(Fig. 3). The area of the scleral part increases continuously with a maximum rate
on day 6 within the first ten postnatal days (Fig. 3A). Starting from day 5 to the
276
O. G. STROEVA AND I. G. PANOVA
10
5
7 9 11
age in days
15
3
5 7 9
age in months
12
Fig. 3. Growth of the eye and its scleral part throughout postnatal life in the rat.
Each point on thefigurewas derived from measurements on 10 to 45 eyes. The total
area of each scleral part was measured by planimetry from photos offlatpreparations
(each point derived from measuring of 10 scleral parts).
Open circles refer to the weight of the whole eye,filledcircles refer to that of the
scleral part, and triangles refer to the area of the scleral part. Vertical lines are
confidence intervals at 95 % significance level.
In square above: (A) the gain of the scleral part area calculated from the Equation
(2), and (B) the gain of the scleral part weight calculated from the Equation (1) (for
details see Methods).
twelfth month of age, the scleral part of the rat eye increases twofold in its weight
and sixfold in its area.
Proliferative activity in the rat RPE resumes during the second postnatal day.
By the end of the second to third days the index of labelled nuclei in the central
zone is about 20% after continuous labelling with [3H]thymidine and then
Retinal pigment epithelium in postnatal rats
7
9
11
13
277
15
Fig. 4. Indices of labelled nuclei at the different RPE zones of the intact eyes after
continuous labelling with [3H]thymidine. (A) Indices of labelled nuclei in the central
zone (filled circles) and at the periphery (open circles, averaged data for four
quadrants), and (B) at the periphery (filled bars) and at the equatorial zones (open
bars): (V), ventral; (N), nasal; (D), dorsal; and (T), temporal; each bar represents
averaged data for the eyes of three animals. Vertical lines are standard error of the
mean.
declines. During days 5-8, the labelling index remains at the 8 % level, then falls
below 5 % by day 9, and below 1 % by the 11-15 days after birth (Fig. 4A). The
average index of labelled nuclei at the RPE periphery was 7 % from the third to
eighth postnatal days, and then dropped (Fig. 4A). No significant difference in
the proportion of DNA-synthesizing cells was observed between different
quadrants of the RPE periphery, whereas in the dorsal and temporal equatorial
zones the number of labelled nuclei exceeded that in the central zone.
Proliferative activity was the same in the nasal and ventral equatorial zones as
in the periphery (Fig. 4B).
Binucleated cells
There are occasional binucleated cells (about 5 %) in the RPE central zone of
newborn rats. Their proportion reaches about 50% in 5-day-old and 80% in
9-day-old rats (Fig. 1). At the periphery the 50 % level of binucleated cells was
observed by day 9 and does not change later (Fig. 5). Thus the RPE zones with
the highest proliferative activity are those with the maximal proportion of
binucleated cells.
278
O. G. STROEVA AND I. G. PANOVA
lOO-i
g 50^
o
v
1 3
5 7
age in days
Fig. 5. The proportion of uninucleated (filled circles) and binucleated (open circles)
cells at the RPE periphery (averaged data for four quadrants of the globe). Each
point derived from cell count on tangential sections of the RPE of one eye (1000 cells
per eye).
100 -i
50 -
2 4
8
20
24
27 30 33
10
h after a single r3H]thymidine injection
Fig. 6. The curve for the fraction of labelled mitoses (FLM) in the central RPE zone
plotted as a function of time following a single pH]thymidine injection given to
4-day-old rats. Mitoticfiguresfrom middle prophase to telophase were scored (30 to
60 mitoses per eye) on tangential sections of the RPE. Each point represents the data
for one eye. Solid line, open circles refer to the FLM at the threshold of not less than
5 grains over nucleus; dotted line,filledcircles refer to the FLM at the threshold of
not less than 15 grains over nucleus.
The cell cycle and origin of binucleated cells
Marshak (1974) was the first to use the cell cycle to show that binucleated cells
are of mitotic origin. However she failed to present any quantitative data against
the idea that binucleated cells could form by a fusion of uninucleated labelled
cells. To dismiss that possibility we have repeated this experiment with 4-day-old
rats using the pulse-chase method of Quastler & Shermann (1959) as well as
double labelling with [3H]thymidine and [14C]thymidine. Grain density over
Retinal pigment epithelium in postnatal rats
279
labelled interphasic RPE nuclei was also determined. The results are documented in Figs 6, 7 and 8.
To construct the curve of labelled mitoses (FLM) initially all mitotic figures
with not less than five grains overlying them were considered as labelled. The
resulting curve gives the mean value of 6-4 h for G2+I/2M, and of 25 h for the
generation time (T) (measured at the 80% level of the FLM curve). The
descending limb of the curve did not fall below the 50 % level which made the
graphic determination of ts impossible (Fig. 6).
No labelled binucleated cells were observed 2 h after [3H]thymidine injection,
and 2-6 % were labelled at 27 h on the FLM curve. The population of labelled
uninucleated cells was homogenous by the criterion of grain density distribution
at 2h (Fig. 7, A, I), but became heterogenous by 27h (Fig. 7, A, II). Nuclei of
distribution of grain
density classes
Fig. 7. Grain density distribution for interphase nuclei of uninucleated and
binucleated cells in the central RPE zone on the 2nd and 27th h of the FLM curve
shown in Fig. 6.
Nuclear area was averaged by measuring from 50 to 70 nuclei of randomly selected
RPE cells on tangential sections; grain density was termed as number of silver grains
per nuclear area. Histograms (A) refer to the grain density over nuclei of
uninucleated cells at 2 h (I), to that of uninucleated cells at 27 h (II), and to that of
binucleated cells at 27 h (III). Histogram (B) refers to the averaged grain density over
each of two nuclei of 100 randomly selected binucleated cells at 27 h. Vertical lines
are confidence interval at 95 % significant level.
280
O. G. STROEVA AND I. G. PANOVA
some cells which had incorporated [3H]thymidine did not divide during this cycle
(e.g. some cells of the fraction of the most heavily labelled nuclei). The population of the least-labelled nuclei increased enormously. These findings could not
be attributed merely to cell division during a single cell cycle, and might indicate
the reutilization of radioactive DNA precursors, similar to that observed for
newts' eyes (Parshina & Mitashov, 1978). No reutilization effect was found when
plotting the curves for labelled mitoses in the neural retina (Denham, 1967;
Stroeva, 1978) and in the neuroepithelia of the iris, but it affected greatly the
pattern of FLM curves for the neuroepithelia of the ciliary body (Stroeva, 1978).
Possibly, it could be related to the barrier function of both pigmented epithelia.
In the histogram showing grain density over the nuclei of binucleated cells at
27 h on the FLM curve (Fig. 7, A, III), the fraction of the most heavily labelled
nuclei was absent, and the fraction of the least-labelled nuclei of binucleated cells
7-
[3H]thymidine
injection
6"
a 5-
1 4-1
8
3-
2-
i.
i
I
1 -
i
[14C]thymidine
injection
9 age in days
Fig. 8. Indices of labelled nuclei of RPE cells after double labelling with p ]
thymidine and [14C]thymidine. Only nuclei of uninucleated RPE cells were initially
labelled with [3H]thymidine on day 3 of age; about 50 % of them became binucleated
by day 4, and the other 50 % became binucleated by postnatal day 5, and then
withdrew from the cell cycle (open bars). Only nuclei of uninucleated cells were
labelled with [14C]thymidine on any day (filled bars). No uninucleated cells with
double label were found. Each bar derived from cell counts in the RPE central zone
on tangential sections, using three animals (1000 cells per eye). Vertical lines
represent the standard error of the mean.
Retinal pigment epithelium in postnatal rats
281
was less than that of uninucleated cells (Fig. 7, A, II). When comparing grain
density over each of the two nuclei of binucleated cells, no statistical differences
were obtained (Fig. 7B). This could not be due to cell fusion with the observed
heterogeneity of labelling of the uninucleated cells at 27 h. The results obtained
unambiguously demonstrate the mitotic origin of binucleated cells.
To avoid the influence of a reutilization process, only labelled mitoses with not
less than 15 grains over them were taken for the FLM curve reconstruction (Fig.
6). With such a correction, the graphic detection of fs at the 50 % level of FLM
curve was possible. It was found to be 15 h (after subtraction of 1 h - the persistence time of [3H]thymidine in the blood).
The duration of the M phase found in the experiment with colchicine (using
Equation (3); see Methods) proved to be 2-3 h (m = 0-7 % ± 0-06; mc = 0-91 %
±0-15; £ = 3h). After appropriate subtraction, tGi and fci were found.
Therefore, the mean T was 25 h, fe was 15-0h, tu was 2-3 h, fci was 2-5 h, and
*G2 was 5-2h.
The number of cell cycles in the RPE between days 3-9 was determined in a
double-labelling experiment. Only nuclei of uninucleated cells were labelled in
the RPE central zone at 2h following the single [3H]thymidine injection. The
indices of labelled nuclei were 3-2 % in the RPE of 3-day-old rats, 5-6 % in that
50
40
30-
20-
10-
7
9
11
age in days
13
15
Fig. 9. Growth of microphthalmic eyes. The growth estimation is based on the
weight data for whole microphthalmic eyes obtained as the result of the lens removal
at the onset of day 2 (filled squares) and at the end of day 4 (open squares), as well
as for whole intact eyes (open circles) and their scleral parts (filled circles). From
three to seven rats were used for each measurement point. Vertical lines represent
the standard error of the mean.
282
O. G. STROEVA AND I. G. PANOVA
B
10
Fig. 10. (A) The left microphthalmic eye of a 15-day-old rat from which the lens had
been removed surgically at the onset of day 2, and (B) the right intact eye from the
same animal.
Fig. 11. A tangential section of the RPE central zone of the intact eye of a 9-day-old
rat. The majority of RPE cells are binucleated ones; at the centre of the
microphotograph a labelled binucleated cell is seen. Melanin grains were bleached
with potassium permanganate. Sections were stained over the emulsion with Carrachi haematoxylin.
Fig. 12. A tangential section from the RPE central zone of the microphthalmic eye
of a 9-day-old rat (the same animal as that shown in Fig. 11). The lens was removed
from the eye at the onset of the 2nd day of age. There are uninucleated cells with
labelled nuclei; at the left a labelled mitosis is seen. Melanin grains were bleached
with potassium permanganate. Section was stained over the emulsion with Carrachi
haematoxylin.
Retinal pigment epithelium in postnatal rats
283
of 4-day-old rats, 6-2 % in the RPE of 5-day-old rats, and thereafter remained
stationary (Fig. 8). In the RPE of 4-day-old rats 50% of labelled cells were
uninucleated and 50 % were binucleated. On the fifth day 92-1 % of all labelled
cells became binucleated. This means that the RPE cell population is
heterogeneous in the duration of the Gi phase. Half the cycling cells had a Gi that
considerably exceeded 5h, the mean time found in the preceding experiment.
Therefore, almost all RPE uninucleated cells which had synthesized DNA at day
3 became binucleated by day 5 and withdrew from the cell cycle. New
uninucleated cells enter the cell cycle during the postnatal days 4-9, as was
revealed by pulse labelling with [14C]thymidine given to the rats as the second
label 2 h before sacrifice. The pulse label with [14C]thymidine was 1-7 ± 0-3 % by
days 4-8 and decreased to 0-7 % by day 9 (Fig. 8). Only two binucleated cells
labelled with [14C]thymidine were seen in the RPE of 6- and 7-day-old rats. No
uninucleated cells with two labels were found. Thus this experiment confirmed
the mitotic origin of binucleated cells and showed that during this period the
majority of RPE cells pass through the cell cycle only once.
Experimental microphthalmia
Eye growth
Lens removal from the rat eye at the onset of day 2 and at the end of day 4
resulted in the same degree of microphthalmia (Figs 9 and 10). The growth
pattern of the intact eyes of the operated animals did not differ from that of
normal animals (cf. Fig. 3 and Fig. 9). The lens removal caused a detachment and
infolding of the neural retina. As a rule the RPE of microphthalmic eyes was
healthy (Figs 11 and 12). A slight increase in nuclear concentration in the RPE
occurred immediately after lens removal and then progressed (Fig. 13). This
g
5-
ca
3
sr 4•g
3
I 2\
a
0
o
2
age in days
Fig. 13. Nuclear concentration in the RPE central zone of microphthalmic (A), and
of intact (B) eyes.
284
0 . G. STROEVA AND I. G. PANOVA
60 4
50-
40*
30 •
•
•
"o
c
•a
_u
20 -
30 "
•
>
\
^\
20-
• \
"3
.2
o 10-
A•
10
<+*
X
t*
a)
•o
C
1
15
15
2 3 5 7
age in days
age in days
•
20-
*
D
20
I
glO
\
B
\
3 5 7
age in days
9
1
l
15
3
5 1 ' 9
age in days
A
K
Fig. 14. Indices of labelled nuclei in the different zones of the RPE of intact and
microphthalmic eyes after continuous labelling with [3H]thymidine.
Labelling indices derived from nuclei count (1000 nuclei per eye at any zone) on
tangential sections from the central RPE zone of the control (A), and microphthalmic eyes (B), as well as at the periphery of the control (C) and of microphthalmic eyes
(D). Each circle represents the data for one eye. In the RPE of microphthalmic eyes
indices of labelled nuclei dropped by day 7 as compared with those of the control.
Asterisks refer to the eyes in which the RPE was invaded by macrophages of vascular
origin (see, Fig. 15).
Retinal pigment epithelium in postnatal rats
285
Fig. 15. The RPE of the microphthalmic eye of 9-day-old rat attacked by
macrophages of vascular origin: monocytes (A) and polymorphonuclear leukocytes
(B), indicated by arrows. Nuclei of all RPE cells are labelled.
means that there is no increase in area of individual RPE cells in microphthalmic
eyes.
Proliferative activity
In the RPE of microphthalmic eyes from which the lens had been removed at
the onset of day 2 the indices of labelled nuclei increased by the end of days 2 and
3, they did not differ statistically by the end of day 5 and dropped in 7-day-old
rats in all RPE zones as compared with those of the control (Fig. 14).
The pattern of cell proliferation in the RPE of some microphthalmic eyes was
affected by two additional factors. One of these was a damage of the RPE as a
result of surgical manipulations. Macrophages of vascular origin invaded the
RPE in such cases, and the number of RPE-labelled nuclei locally increased
enormously (Fig. 15). Migration of some RPE cells from the epithelial layer into
the subretinal space, and their transformation into macrophage-like elements
was another event affecting the labelling indices in some microphthalmic eyes,
as the mitotic division of the RPE cells had preceded such a migration.
Binucleated cells
The eye growth suppression also affected the proportion of binucleated cells.
By the end of the fifth postnatal day the proportion of binucleated cells reached
50 % in the central RPE zone of the microphthalmic eyes, just as in control
eyes, and then remained stationary (Fig. 16A, B). A considerable decrease in
the final proportion of binucleated cells was also noted at the RPE periphery
(Fig. 16C, D). As a result, in 9- and 15-day-old rats the proportion of
binucleated cells in the RPE of the microphthalmic eyes was about 30 % less
than in the control.
286
O. G. STROEVA AND I. G. PANOVA
2 3
5
7
9
* 15
2 3
age in days
100 1
100
50-
50
llJL
9
5
7
9
age in days
15
D
9
15
age in days
age in days
Fig. 16. Proportion of uninucleated and binucleated cells in the different RPE zones
of intact and microphthalmic eyes (the same samples as in Fig. 14).
The percentage of cells of both types is based on counting at least 1000 cells per
zone for each eye. The proportion of uninucleated cells (rilled circles) and
binucleated cells (open circles) was obtained for the central zone of the intact eyes
(A) and microphthalmic eyes (B) (each circle represents the data for one eye), as well
as for the periphery of the control (C), and of the microphthalmic eyes (D): open bars
refer to uninucleated cells and filled bars refer to binucleated ones (each bar
represents averaged data for three tofiveeyes). The proportion of the uninucleated
and binucleated cells in the RPE of the microphthalmic eyes does not change after
postnatal day 5.
Size of nuclei
No cells with large nuclei typical for the control (Fig. 17A, B, C) were present
in the RPE of microphthalmic eyes of 9- and 15-day-old rats (Fig. 17D, E).
Earlier such cells were shown to be polyploid by use of spectrophotometry
(Marshak & Stroeva, 1973, 1974). The total size of RPE cell nuclei was also
decreased (Figs 17 and 18). This phenomenon will be discussed in detail
elsewhere.
Retinal pigment epithelium in postnatal rats
287
40
20
40
20
40
20
D
40
20
40
20
80 120 ie
f~...
nuclear area
Fig. 17. Area size distribution for nuclei of RPE cells in the central zone of the intact
(A, B, C) and the microphthalmic (D, E) eyes: of 1-day-old rats (A), of 9-day-old
rats (B and D), and of 15-day-old rats (C and E) (the same samples as in Figs 13,14
and 15). Nuclear area was calculated as that of an ellipse using 50 to 70 nuclei per eye.
Each histogram represents averaged data for three eyes. In the RPE of microphthalmic eyes the cells with large nuclei seen in that of the intact eyes were absent. The
total nuclear area in the RPE of the microphthalmic eyes was also diminished as
compared with that of intact eyes (see also, Fig. 18).
Table 1. Indices of labelled nuclei in the central zone of the RPE of intact and
microphthalmic eyes from 5-day-old rats
Percentage of labelled nuclei in the central zone of the RPE after
continuous labelling with [3]thymidine
Number of
animal
of right intact eye
of left microphthalmic eye"
10-1
8-1
5-7
9-2
7-4
* microphthalmia was induced by lens removal at the end of day 4.
** the RPE was invaded by macrophages of vascular origin.
8-3**
4-5
2-2
6-0**
2-4
288
O. G. STROEVA AND I. G. PANOVA
Fig. 18. Microphotograph of RPE cells from the central zone of the microphthalmic
eye of-a 15-day-old rat (the same sample as in Fig. 10A). Melanin grains were
decoloured with potassium permanganate. The section was stained with Carrachi
haematoxylin. The total size of nuclei is diminished as compared with that of the
control (cf. Figs 11 and 17).
Lag period
The inhibitory effect of eye growth suppression on RPE cell proliferation, as
a result of lens removal at the onset of day 2, did not manifest itself up to
postnatal day 5 (Figs 14 and 16). If, however, the lens was extirpated at the end
of day 4 the proportion of labelled nuclei decreased as early as by the first
postoperative day (Table 1). Therefore, the period of RPE cell proliferation
from postnatal days 2-5 is a peculiar one and independent of intraocular
pressure.
DISCUSSION
The data obtained clearly showed that in the rat RPE the binucleated cells are
formed after birth as a result of the postnatal wave of acytokinetic mitoses. The
great majority of uninucleated cells pass through the cell cycle only once and
become binucleated. Few uninucleated and binucleated cells enter the cell cycle
more than once, but do not pass through mitosis, and form the fraction of
polyploid cells. Since the proportion of uninucleated and binucleated cells does
not change after day 9 (Fig. 1), the last portion of uninucleated cells destined to
become binucleated has to enter the cell cycle not later than day 8. However,
DNA-synthesizing cells can be seen at least up to day 15. Probably these cells
become polyploid and persist as a Gi subpopulation of the RPE.
The formation of a considerable proportion of binucleated and polyploid cells
can be experimentally avoided by factors which prevent cell passage from G\ to
Retinal pigment epithelium in postnatal rats
289
S phase (in our case by removing the intraocular pressure). The phenomenon
seems to be a general one. Thus lens removal from the chick embryo eye at the
fifth day of incubation results in a seven- to tenfold decrease in DNAsynthesizing cells in the RPE compared with intact eyes (Stroeva, Akhabadze,
Lobacheva & Panova, 1980). In rats the intraocular pressure begins to act as a
factor in RPE growth by day 5. The drop in the number of DNA-synthesizing
cells in the RPE of microphthalmic eyes can be explained by the in vitro experiments in which stretching of cells was shown to stimulate their entry into the S
phase. (Folkman & Moscona, 1978; Curtis & Seehar, 1978).
The stretching effect of intraocular pressure on RPE cell proliferation in the
eye in situ might be modified by additional factors. It seems that the proliferation
between days 2-5 in rats may be one such modification. Nevertheless, the effect
of intraocular pressure on the RPE cell population seems to persist throughout
the life of the animals. For instance, in the microphthalmic eyes of adult mutant
MSUBI rats the suppression of eye growth affects mainly the formation of multinucleated cells essential for the RPE of old animals (Stroeva & Nikiphorovskaya, 1970). Therefore, the regulation of RPE growth by intraocular pressure
involves not only the increase in cell size through mechanical tension
(Coulombre, 1956; Coulombre, Steinberg & Coulombre, 1963), but the control
of the number of cycling cells as well. It seems that the same regularity also holds
for the sclera (Coulombre & Herrmann, 1965).
The study of RPE proliferation in the rat revealed three facts of interest which
called our attention to the period of RPE development between days 2-5. First,
cell proliferation during this time is independent of intraocular pressure.
Therefore, the postnatal wave of proliferation is not initiated by growth of the
scleral part. It seems more probable that it results from earlier influences on the
RPE which overlap the inhibitory effects of intraocular pressure removal.
Second, the pattern of postnatal cell proliferation is opposite to that during the
intrauterine life, when the RPE grows mainly at the expense of cell divisions at
the marginal zones of the eye (Zavarsin& Stroeva, 1964). After birth the number
of cells remains constant, and the RPE area increases mainly in the central zone
due to the process of cell binucleation (the area of one binucleated cell is
approximately l-6x as large as that of a uninucleated cell (Marshak, Stroeva &
Brodsky, 1976). Third, the cell cycle of RPE cells at this time is characterized by
a relatively lengthy Gi phase (about 20 % of total cycle time in comparison with
4-2 to 7-8 % in the iris and ciliary body) and a short G\ phase (10 % compared
with 24-0 to 45-5% in the neuroepithelia of the anterior complex of the eye
(Stroeva, 1978). It is of interest in the light of these findings that in cultured cells
of mouse melanomas the Gi phase is a target for differentiate action of
melanotropic hormones which induce additional melanin synthesis (Varga et al.
1974). If the same is true for normal pigment cells, the RPE should be most
sensitive to the differentiative effect of melanotropins at the time when RPE cell
proliferation is highest. Moreover, this stage in normal animals should be a
EMB75
290
O. G. STROEVA AND I. G. PANOVA
starting point for a new wave of melanogenesis in the RPE. On the basis of these
findings we assume that postnatal proliferation might be of importance for the
final RPE melanotic differentiation. It has been indeed demonstrated (Stroeva
& Bibikova, 1982) that stimulation of melanin synthesis above the basic level by
ACTH in RPE cells in organ culture was possible only in the eyes of 3-day-old
rats. It has been also shown that postnatal melanization in rat RPE is realized in
two phases. Synthetic processes preceding the second phase of melanin accumulation in the RPE in situ begin after the third day, and a new wave of melanin
synthesis is realized after the fifth day against a background of lower cell
proliferation (Stroeva, Panova, Poplinskaya & Bibikova, 1982). These data are
consistent with the hypothesis proposed above.
The formation of numerous binucleated cells in the rat RPE can be considered
in terms of this hypothesis. If the postnatal RPE cell proliferation is necessary
mainly for differentiative action of melanotropins it might be excessive in respect
to growth of the RPE area. The RPE area cannot exceed that of scleral part,
whose size is regulated by the intraocular pressure. Thus the binucleation of RPE
cells could be considered a useful answer to a difficult problem. Indeed, the area
of one binucleated cell is about three quarters of two uninucleated ones. This
enables RPE cells to pass through the cell cycle and provides a moderate increase
in the RPE area at the same time. The delay of some RPE cells in Gi might also
be considered as an additional means to the same end. If so, the formation of
binucleated cells in the RPE might be indicative of excessive cell proliferation
stimulated by factors additional to the tension of the eye walls.
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(Accepted 10 January 1983)