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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. 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