Changes in DNA, RNA, and protein synthesis in the developing lens

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1 Changes in DNA, RNA, and protein synthesis in the developing lens Calvin Hanna Lens cell DNA, RNA, and protein synthesis in the developing mouse eye were studied with the use of tritium-labeled thymidine, uridine, and l-leucine and autoradiographic techniques. In the mouse embryonic lens, epithelial cells undergoing DNA synthesis were found over the entire anterior lens surface. From birth and until the eyes opened the percentage of epithelial cells undergoing DNA synthesis rapidly decreased. Later the percentage of epithelial cells undergoing DNA synthesis teas nearly constant as the germinative zone became localized in the lens equator region. RNA synthesis occurred in all nucleated cells of the developing lens from the embryonic stage until the eyelids opened. With lens maturity the S H uridine was incorporated into the RNA of only the more superficial cells. A similar pattern of tritium incorporation was seen with S H l-leucine. he use of tritium-labeled biochemical intermediates together with the autoradiographic technique is a powerful tool for the study of the physiological processes of a cell. This technique is especially helpful when only a few cells of a type are involved or where mixed cell populations are to be studied. Studies on the lens with the use of autoradiographic techniques have centered around 3 H-thymidine, an intermediate in deoxyribonucleic acid (DNA) synthesis. 3 H-thymidine is incorporated into the DNA of the lens epithelial cell during the synthetic stages of cell division. Use was made of this fact in two early reports on 3 H-thymidine uptake by lens epithelial cells. Harding, Hughes, Bond, and Schork 1 incubated the lenses from rats, frogs, and young rabbits in Eagle's basal medium con- From the Department of Pharmacology, University of Arkansas Medical Center, Little Rock, Ark. This investigation was supported by PHS Grants NiB and NB from' the National Institute of Neurological Diseases and Blindness, United States Public Health Service. 480 taining 3 H-thymidine. The lens capsule with epithelial cells attached was removed and placed on a glass slide (flat mount) and covered with photographic emulsion. An analysis of the resulting autoradiogram revealed tritium-labeled epithelial cells in a band that corresponded to the germinative zone of the lens which is just anterior to the lens equator. In another study, the rabbit lens was wounded with a small needle and one day later the 3 H-thymidine was injected into the anterior chamber. 2 This study revealed that epithelial cells around the wound edge incorporated 3 H- thymidine. Since that time a number of studies have been carried out on the details of the 3 H-thymidine incorporation into the epithelial cells after lens wounding or manipulation. 3 " 5 Additional studies on the incorporation of 3 H-thymidine into lens epithelial cells have been carried out on various species and under various conditions. Hanna and O'Brien, 0 found that 3 H-thymidine was incorporated into the lens epithelial cells located in the germinative zone of the

2 Volume 4 Number 4 Changes in DNA, RNA, and 'protein synthesis 481 adult rat lens. In addition, it was found that these tritium-labeled epithelial cells migrated posteriorly at a slow rate. In this same study the rats fed a galactose diet for 12 days exhibited a seventeenfold increase in the number of cells incorporating 3 H- thymidine between the fourth and sixth days. During this time the germinative zone spread over the entire central area of the lens and there was a corresponding increase in number of cells incorporating 3 H-thymidine in the iris and ciliary processes. The influence of the age of the rat on the lens epithelial cell incorporation of 3 H-thymidme was studied by Hanna and O'Brien. 7 It was found that the rate of proliferation and migration of the epithelial cells was directly related to age. Brolin, Diderholmh, and Hammar 8 also utilized 3 H-thymidine to estimate the migration of lens epithelial cells. The mouse and dog lenses were found to have a germinative zone very similar to that of the rat. 9 In the human the 3 H-thymidine was found to be incorporated into epithelial cells in a similar spatial pattern in the lenses of a teen-age child, a 35-year-old woman, and in the extracted lenses from 7 elderly people with senile cataracts. Cell division is composed of four major stages each of which can be determined in most cases with the use of 3 H-thymidine. For this determination the 3 H-thymidine is brought into contact with the dividing cells and the cells are killed at various times later. The cellular tritium uptake is analyzed autoradiographically. By calculating the per cent of tritium-labeled cells at one hour and the per cent of tritiumlabeled mitotic figures at intervals thereafter, it is possible to estimate the following: The period of DNA synthesis (S phase), the premitotic interval (G 2 phase), the period of mitosis (M phase), and the postmitotic gap (G :l phase). The cell cycle has been estimated for a number of tissues including the cornea and lens (reviewed by Bertalanffy 10 ). Thomson, Pirie, and Overall 11 attempted to do this with a small group of 3- to 4-month-old rabbits. As pointed out by the authors, the resulting calculations are at best approximate because one animal was used at a critical time period necessary in the calculations. Mikulicich and Young 12 used an adequate number of 35-day-old rats and arrived at an estimated cell cycle in the germinative zone as follows: S phase lovz hours, G 2 phase minimum 2Vz hours, M phase between 2 and 5V2 hours, and G x phase of about 36 hours. Scullica, Grimes, and Mc- Elvain 13 ' 14 similarly studied several stages of cell division in the 28- to 32-week-old rat. They estimated the following cycle: S phase 10 hours, G 2 phase 5 hours, M phase 1% hours, G t phase 19 days (in the germinative zone). These authors also studied the effects of ionizing radiation on the epithelial cells of the rat lens. They found, as did Hanna and O'Brien 15 ' 1G an initial inhibition followed by an overs wing in the number of lens epithelial cells undergoing DNA synthesis. Also, these epithelial cells moved a short distance before many of the cells disintegrated with a loss of the tritium-labeled DNA. Experimental Various species of animals were injected into the anterior chamber with 5 nc of tritium-labeled compound, 1 me. per milliliter, through a 33 gauge needle. Most of the animals were anesthetized with ether and topical lidocaine. The animals were usually killed 2 hours later and the enucleated eyes fixed in formalin or Carnoy's solution. The formalin-fixed tissues were paraffin embedded and cut into 5 /* thick sagittal plane sections. The capsule and epithelial cells of the Carnoy's fixed lenses were flat mounted onto glass slides. Both tissue preparations were treated with xylene and hydrated through the alcohols, washed in cold water, and painted with Kodak NTB3 liquid emulsion. After exposure for 10 to 60 days in the dark the slides were developed in Kodak D-19 developer, cleared with Kodak acid fixer, and stained with hematoxylin and eosin. 35 In studies on the developing mouse lens the tritium-labeled compounds were injected intraperitoneally. The tritium-labeled compounds 0 used are as follows: adenosine (3 c. per millimole [c./mm.]), cytidine These compounds were purchased from the following sources: New England Nuclear Corp., Boston, Mass.; Nuclear-Chicago Corp., Des Plaines, 111.; Schwarz Bio- Research, Inc., Orangeburg, N. J.; Volk Radiochemical Co., Skokie, 111.

3 482 Hunna Investigative Ophthalmology August 1965 (3 c./mm.), cytosine arabinoside (3 c./mm.), deoxyadenosine (2.8 c./mm.), deoxycytidine (3 c./mm.), deoxyguanosine (2.8 c./mm.), deoxyuridine (1.3 c/ram.), Z-histidine (1.7 c./mm.), hypoxanthine (1.7 c./mm.), 5-iodo-2'-deoxyuridine (-6-3 H, 0.34 c./mm.), Meucine (10 c./mm.), orotic acid (-5-3 H, 0.1 c./mm.), Z-phenylalanine (1.6 c./mm.), Z-proline (3.2 c./mm.), thymidine (0.8 to 8.8 c./mm.), and uridine (-5-3 H, 1.7 to 13 c./mm., and -5,6-3 H, 1.7 c./mm.). Selected sections or flat mounts of lenses from the various studies were treated with DNase or RNase. 017 Unless otherwise indicated, the tritium-labeled intermediates that gave only a nuclear label, or a nuclear label at 45 minutes and a nuclear-cytoplasmic label at 2 hours represented DNA and RNA labeling, respectively. GERMINATIVE ZONE NNULAR PAD Fig. 1. Schematic representation of chicken lens. The dark arrows in the annular pad region refer to the path taken by epithelial cells through the pad and the number of weeks to arrive at each point. The cross-hatched lines above the cell nuclei indicate the cells undergoing RNA synthesis as determined by 3 H-uridine. DNA metabolism The germinative zone of the lens of adult animals was located as a band of epithelial cells undergoing both mitosis and DNA synthesis. The germinative zone in the reptilian type of lens was located in a narrow band of epithelial cells just anterior to the annular pad (Fig. 1). This type of eye was found to include the following: the Lacertilian eye, the chameleon (Sauropsida); the Crocodilian eye, the 6- Worthington Biochemical Co., Freehold, N. J. month-old American alligator (Caiman); and the avian eye, the chicken (Gallus domesticus, Leghorn). A narrow germinative zone like that in the human eye, i.e., located just anterior to the lens equator and across from the ciliary processes (Fig. 2) was found in the following types of eyes: the Anuran eye, the frog (Rana pipiens); the Placentalian eye, the rabbit (Rodentia, Leporidae, Oryctolagus); the rat and the mouse (Rodentia, Muridae, Rattus, and Muss); the cat and the dog (Carnivora, Felis and Canis) and the human. Two exceptions were noted to this general classification of the germinative zone. The lens of the guinea pig (Caviodea, Caviidae) exhibited a broad germinative zone s while the minute lens from the American short-tailed shrew (Blarina brevicauda) had no detectable germinative zone. The cell cycle of the developing lens of the Swiss mouse was determined with 3 H- thymidine. Animals 3, 6, 12, and 24 days old were injected intraperitoneally with 1 me. per kilogram of 3 H-thymidine. The mice were killed in the afternoons in the summer time in groups of 6 at 1, 2, 5, 7, 12, and 17 hours later. Half of the eyes were fixed in Carnoy's and the lens epithelial layer flat mounted, and half of the eyes were fixed in formalin and the lenses cut through the sagittal plane. The center of the germinative zone was located in the flat mount preparations and the per cent of cells undergoing DNA synthesis and the per cent of tritium-labeled mitotic figures were determined at each time interval. From a plot of these values versus time the following phases of cell division were calculated 18 : G x phase, AQVz hours; S phase, 9 hours; G 2 phase, 2V2 to 5 hours; M phase, W2 to 4 hours; with a cell cycle of 56 hours (Fig. 2). No marked differences were found in the cell cycle for the varying ages of the mice. The spatial distribution of the cells undergoing DNA synthesis was determined on the sagittal plane sections and the results are given in Fig. 2. In addition to the ages of the mice given

4 Volume 4 Ntimber 4 Changes in DNA, RNA, and protein synthesis 483 Fig. 2. Graphic representation of the number and the average position of lens epithelial cells undergoing DNA synthesis in mice of varying days (d) of age. The semicircle represents half a sagittal plane section of a mouse lens with a nuclear bow region. The black dots represent the average position of epitlielial cells undergoing DNA synthesis while the cross-hatched area represents the range of cells found in DNA synthesis. The point at which epithelial cells become lens fibers is indicated by a dash and each 10 cells anterior to this are indicated by a dash. The circle in the lower right-hand corner represents the length of a cell cycle (56 hours) and the percentage of the total cycle taken up by the various phases of division. in Fig. 2 those of the 2 day prenatal and of the 1 and 180 day postnatal mice were studied. DNA synthesis occurred in about 1 epithelial cell in 8 in the 2 day prenatal lens. The germinative zone in the 1 day postnatal mouse involved most of the epithelial cells except those in the premeridional row region near the lens equator. This pattern of DNA synthesis continued with a slight decrease in the per cent of S phase epithelial cells until the eyelids began to open at 12 days of age. After this time the germinative zone narrowed and centered into a region just anterior to the lens equator (180-day-old mouse). These results on the spatial distribution of S phase epithelial cells are similar to the results reported by Hanna and O'Brien 7 on the developing rat lens. The mouse lens develops considerably faster than does the human lens and the lens development in the mouse is in the embryonic stage until after birth. Therefore it is possible to study a number of the developmental changes in the lens of the mouse after birth. The developing mouse lens from about 12 days after conception until the eyelids open at about 12 days of age corresponds to the period in the human from the time when the lens disc thickens until birth. In the mouse the lens vesicle closes at 11 days and by 12% days the lens vesicle detaches from the surface epithelium and the lens capsule begins to form. The posterior cells of the lens vesicle grow and obliterate the lens cavity by 13 days. From 13 days until birth the nuclei of the primary fibers begin to disappear to form the fetal nucleus which is then covered by secondaiy lens fibers. The newborn mouse lens, in development, is equivalent to the 30 to 35 mm. long human embiyo after which the human fetal period begins. 19 The S phase and the M phase of cell division were found to dissociate in the 12-day-old mouse lens. The S phase in this lens (2 hour 3 H-thymidine exposure) ex-

5 484 Hanna Investigative Ophthalmology August 1965 tended from the meridional row cells to the central area of the lens while the M phase cells were in a narrow zone just anterior to the meridional row cells. In the zone of M phase cells, tritium-labeled mitotic figures were found 5, 7, and 9 hours after the 3 H-thymidine injection. Only a rare S phase cell in the central area of the lens underwent mitosis by 17 hours after the 3 H-thymidine injection. These results suggested that some of the epithelial cells in the central area of the lens were arrested in the G 2 phase of cell division. Evidence has been presented to show that the epithelial cells of the mouse are arrested in the G 2 phase of cell division." Results obtained after lens wounding may indicate a similar arrest of cells in the G 2 phase. Rats and rabbits were wounded with a thin needle. 2 3 H-thymidine was injected into the anterior chamber 18, 24, 30, and 36 hours after wounding and the animals killed 2 hours later. At 18 hours the epithelial cells in the S phase were located near the wound edge. By 24 hours the S phase cells were located about halfway between the wound edge and the lens equator and M phase cells were located just anterior to this. The M phase cells were intermingled with the S phase cells and the mitotic figure did not occur synchronously. This sequence of events is clearly presented in the photomicrographs of Harding, Feldherr, and Srinivasan. 20 By 30 hours a second zone of S phase cells appeared at the wound edge; however, by 36 hours no second zone of M phase cells appeared. These results suggest that some of the epithelial cells in the central area of the lens had been arrested in the G 2 phase of cell division and that after wounding more cells were arrested in the G 2 phase. A number of authors have noted the migration of lens epithelial cells by utilizing 3 H-thymidine and autoradiography. In each case a 2- to 4-month-old rat or rabbit was studied for about 2 weeks. 11 " 14 This migration of lens epithelial cells into the lens cortex and the formation of lens fibers have been studied in detail by Brolin, Diderholmh, and Hammar s and by Hanna and O'Brien. 0 ' 7 In the very young rat the epithelial cells migrate from the germinative zone and into the lens cortex to the point where the lens fiber nucleus is lost in about 8 weeks. By the time the rat is 5 months old this migration and differentiation into nonnucleated lens fibers take at least 8 months and the migration of these cells in the adult rat takes considerably longer. 7 A parallel exists between the rate at which lens fibers develop and the rate at which an ionizing radiation cataract develops. 15 ' 10 This parallelism has not been established for the reptilian eye since this eye is very resistant to ionizing radiation. In a cold-blooded reptile, the young (6 months) American alligator, a dose of 10,000 r of gamma rays from C0 Co was used without apparent effect on the transparency of the lens over a 6 month period. A warmblooded animal with a reptilian type of eye, the chicken, is also resistant to ionizing radiation. Pirie 21 ' 22 irradiated the chick embryo (12 day) and adult chicken with up to 6,000 r of x-rays and a cataract was not observed in 6 months. It is possible that the 12 day embryo is at a developmental stage that is resistant to cataract development, 23 but it is more difficult to understand why the adult hen did not develop cataracts. In the chicken lens many epithelial cells are held in the annular pad compared to the few epithelial cells in the meridional rows in the rat lens, and it is possible that x-ray-damaged chicken epithelial cells reach the lens cortex much later. Six-week-old male Leghorn chickens were injected into the anterior chamber with 3 H-thymidine and then killed 1, 2, 7, 14, 18, and 30 weeks later. Some of the chickens at 14 weeks were given a second injection of 3 H-thymidine. In the lenses the germinative zone cells migrated into the annular pad at a slow rate as indicated in Fig. 1. The second injection of 3 H-thymidine into the 14-week-old chicken revealed a 20 per cent reduction in the rate of lens epithelial cell migration compared to that

6 Volume 4 Number 4 Changes in DNA, RNA, and protein synthesis 485 of the 6-week-old chicken. It is roughly estimated that the transient time of epithelial cells through the annular pad is on the order of 18 months. Further, the chicken lens was found to grow from the fifth week on but the number of cells in the annular pad decreased with time. Thus the apparent rate of movement of epithelial cells and the estimated time for the transit of epithelial cells through the annular pad can only be roughly estimated. In any event, the time it takes for a chick epithelial cell to become a lens fiber is many times longer than that for the young rat or rabbit. Quite possibly the chicken lens should be observed over a several-year period before it is concluded whether or not radiation cataracts can be produced. Several types of assault will induce the lens epithelial cells to undergo a change in DNA synthesis. Galactose-fed rats, for example, were found to exhibit a marked increase in the number of epithelial cells undergoing DNA synthesis. 0 Lens wounding will produce a similar effect. 2 In both of these examples the epithelial cells in the meridional rows do not undergo DNA synthesis. A study of the effects of cornea freezing revealed an. interesting phenomenon. Rabbit corneas were frozen with diy ice for 1 minute and 24 hours later 'H-thymidine was injected into the anterior chamber. Sections of the eyes revealed that lens epithelial cells in the meridional rows and nucleated fibers in the nuclear lens bow (superficial) underwent DNA synthesis. This indicated that the developing lens fibers are capable of undergoing DNA synthesis although these fibers did not change shape during this process, nor was the transparency of the lens altered during the short observation period. RNA metabolism A study of the incorporation of 5-3 H uridine (15 me. per milligram intraperitoneally) was carried out in 1-, 3-, 6-, 12-, 24-, 96-, and 180-day-old Swiss mice. The mice were killed 2 hours, 1, 3, 7, 14, and 12 days later in groups of three. This dose of 3 H-uridine intensely labeled the RNA of heart, liver, lung, and kidney cells while the RNA in cells of the eye was minimally labeled. In none of the mice studied was tritium-labeled RNA found in lens cells 7, 14, or 21 days after the injection of 3 H-uridine. Almost all nucleated cells of the lens contained tritium-labeled RNA in the 1- and 3-day-old mice 2 hours after the injection of 3 H-uridine. In these lens cells the tritium was localized in or around the cell nucleus. By 1 and 3 days after the 3 H-uridine injections the tritiumlabeled RNA was found throughout the cell and the grain count per cell was much reduced at 3 days. In mice older than 3 days of age, the tritium-labeled RNA was found less often in the more mature lens fibers of the cortex as the age of the animal increased. The tritium-labeled RNA was found only in the epithelial cells and in the most superficial lens fibers of mice older than 24 days of age (Fig. 3). In the adult mouse, rat, rabbit, and senile cataractous human lenses after 2 hours' exposure to 3 H-uridine, the tritium-labeled RNA was found in all epithelial cells and in only the newly forming fibers in the lens equator region (Fig. 3). The pattern of RNA synthesis in the lens epithelial cells was studied in lens flat mount preparations of the rat and human. The lenses were incubated in Eagle's basal medium containing 3 H-uridine for 2 hours. It was found that the epithelial cells over the central area of the lens were undergoing a uniform amount of RNA synthesis (Fig. 4). The cells in the germinative zone were synthesizing a similar amount of RNA, but the cells in the meridional rows were undergoing a reduced amount of RNA synthesis. The newly forming fibers at the edge of the lens epithelium, however, were undergoing a high rate of RNA synthesis. This fact is graphically illustrated in Fig. 4 where 3 H-leucine was utilized but the results were similar to those obtained with 3 H-uridine. One day after lens wounding there were a large number of epithelial cells in the

7 486 Banna EPITHELIUM Fig. 3. Graphic representation of RNA metabolism in the lens of an adult rat eye. Two hours after the injection, into the anterior chamber of 5-3Huridine, the tritium label was found in and around the nuclei of cells covered by the cross-hatching. By 1 day the tritium was found throughout these same cells with the highest concentration in the newly developing lens fibers as indicated by the dark cross-hatching in the equator region. Investigative Ophthalmology August 1965 M phase of cell division. Flat mount preparations of these lenses were found useful in the study of RNA synthesis in relation to the mitotic stages. 3H-uridine was injected into the anterior chamber of rat eye given a lens wound 1 day before. Lenses were removed 45 minutes and 2 hours later. An examination of the flat mount lens preparation revealed that about half of the anaphase and metaphase cells were undergoing RNA synthesis at 45 minutes, whereas all mitotic phases contained tritium-labeled RNA at 2 hours. Therefore, RNA synthesis in the lens epithelial cell is a continuous process except for a short interval during anaphase and metaphase. A comparison was made of the incorporation of 3H-uridine into the RNA of the various cells of the eye. A dose of 30 me. per kilogram of 3H-uridine (13 c./mm.) was injected intraperitoneally into 24-dayold mice. The mice were killed 2 hours Fig. 4. Autoradiograph of a flat mount preparation of epithelial cells from a senile cataract human lens after 2 hours' incubation in Eagle's basal medium containing 5-3H-uridi.ne. The black dots and area over and around each cell nucleus are an indication of the extent of RNA synthesis in these cells. Note the nearly uniform amount of RNA synthesis in this central area of the lens epithelium. (x400; reduced %.)

8 Volume 4 Number 4 Changes in DNA, RNA, and protein synthesis 4S7 later and the eyes prepared for autoradiographic analysis. The exposed silver grains in the photographic emulsion over individual cells were counted and averaged. The results of this study are given in Table I. It can be seen from the table that the developing lens fibers incorporated the greatest amount of 3 H-uridine into RNA of all cells in the globe of the eye. Autoradiograms of cells from the liver, heart, spleen, and kidneys taken from these same animals contained so much tritium-labeled RNA that the emulsion was overexposed after a time of exposure equivalent to that used for the eye tissues. In the adult mouse, rat, rabbit, and human lens the penetration of 3 H-uridine into the older lens cortex fibers was too slow to obtain information on the RNA synthesis of lens fibers nuclear extrusion. The young chicken (8 weeks) was found to be very useful for this study because the 3 H-uridine Table I. Grain count over individual cells of various parts of the mouse eye (24 days old) 2 hours after the intraperitoneal injection of 3 H-uridine or 3 H-leucine (average of 4 eyes each) Area of eye Lens: Epithelium, central equator Cortex, equator anterior posterior Nucleus Cornea: Epithelium Stroma Endothelium Iris Ciliary processes Eyelid Coroid Ocular muscle Retina: Ganglion cells Inner nuclear layer cells Uridine- RNA Leucineprotein readily penetrated into these lenses. To establish the diffusion of 3 H-uridine into the chicken lens, the tritiated compound was injected into the anterior chamber with the least possible mixing. The lens taken at 2 hours showed a much greater intensity of tritium-labeled RNA in the anterior half of the lens than in the posterior half. This information was of importance because the nucleus of lens fibers in the process of nuclear extrusion was located in the anterior half of the lens. These cortex fibers in the process of differentiating into nuclear fibers were found in several progressive stages of nuclear disintegration. An early stage was located with a loss of nuclear granular stain. At a later stage about one-half of the nucleus did not stain and further toward the lens nucleus the fibers were found with a small, darkly staining mass in place of a nucleus. None of these cells were found to be undergoing a measurable amount of RNA synthesis. Protein metabolism Several amino acids with high specific activity and stability of tritium label are available for studies on protein synthesis. Of these 4,5-3 H-Z-leucine was especially useful because this amino acid is generally incorporated into proteins. 24 Four amino acids were chosen for this study. Leucine and 3 H-ring-Z-phenylalanine were chosen as representative of the more hydrotropic amino acids. Levohistidine ( 3 H) and 5-3 H- J-proline were chosen as representative of the more lipotropic amino acids. However, histidine and praline are to some extent converted into other compounds besides being incorporated into protein. The amino acids were injected into the anterior chamber of rats and the animals were killed 2 hours later. The tritium label when found was present in all parts of the nucleated cell. Leucine ( 3 H) and 3 H-phenylalanine gave a similar pattern of lens protein incorporation in the epithelial and cortex fiber cells (Figs. 5 and 6 and Table I). The distribution of tritium from these

9 Inoestigatioe Ophthalmology August Hanna Fig. 5. Autoradiograph of lens epithelial cell from a flat mount preparation of rat lens removed 2 hours after the anterior chamber injection of 3H-leucine. The slide was destained and therefore the intensity and frequency of the black dots give an indication of the rate of protein synthesis in these cells. The right-hand side of the figure indicates a high rate of protein synthesis in a narrow band of epithelial cells differentiating into lens fibers. (x300; reduced V&.) EPITHELIUM, GERMINATIVE ZONE CAPSULE Fig. 6. Graphic representation of protein synthesis in adult rat lenses. Cross-hatched area represents protein synthesis 2 hours after the injection into the anterior chamber of 3H-leucine. When 3 Hhistidine and 8H-proline were used, the indication of protein incorporation of these two amino acids is indicated by the heavy cross-hatched lines. The lens capsule above the epithelial cells was found to contain a small amount of tritium also. two amino acids was very similar to that obtained with 3H-uridine. The developing mouse lens was studied in a manner similar to that for 3H-uridine except SH-Ieucine (10 me. per kilogram) was used. The results obtained with 3H-leucine were very similar to those with 3H-uridine except proteins were labeled in nucleated cells of the lens of mice. In general, protein synthesis occurred in those cells that were also undergoing RNA synthesis. Histidine ( 3 H) and 3H-proline gave results similar to those of Ieucine on the rat lens except these 2 amino acids were incorporated into the protein of the newly developing lens fibers to a much greater extent (Fig. 7). Also, ah-histidine was incorporated into protein over the posterior part of the lens at 2 hours while 3H-proline was intensely incorporated into the ganglion cells of the retina.

10 Volume 4 Number 4 Changes in DNA, RNA, and protein synthesis 4S9 Fig. 7. Autoracliogram of sagittal plane the anterior chamber of 3H-histidine. indicated by the blackness of the area. of the autoradiograph since the area reduced %.) lens section of rat killed 2 hours after an injection into The rate of protein incorporation of 3H-histidine is A torn lens section was chosen to show the resolution without tissue underneath it is almost clear. (x250; Applications The potential of lens epithelial cells to utilize various metabolic intermediates and related compounds was studied in the rat and human lens. Adult rats were injected in the anterior chamber with various tritium-labeled compounds and the animals were killed 45 minutes and 2 hours later. The lenses were sectioned or made" into flat mount preparations for autoradiographic analysis. With the compounds studied four general types of autoradiographic grain patterns were obtained: (1) The grains were located directly over the cell nucleus of epithelial cells in the germinative zone. This DNA pattern was produced by tritium-labeled thymidine, IDU, deoxyuridine, deoxycytidine, deoxyadenosine. (2) The grains were directly over the nucleus at 45 minutes and over the entire cell at 2 hours. This RNA pattern was found over most nucleated cells of the lens and it was obtained with tritium-labeled 5-8H-uridine, and 5-8H-orotic acid. (3) The grains were over the entire cell at 45 minutes and at 2 hours. The protein type of labeling pattern was produced by tritiumlabeled leucine, phenylalanine, histidine, and proline. (4) A mixed RNA and DNA grain pattern was found with tritiumlabeled cytidine, adenosine, guanosine, 5,63 H-uridine, 8-3H-hypoxanthine and deoxyguanosine. The deoxyguanosine in aqueous solution was stored in a frozen state and it is possible that some decomposition occurred in preparing the material for injection. A similar pattern of tritium-labeled cells occurred with these compounds in the cells of the cornea. A similar study was carried out on the human senile cataract lens except the lens was incubated in Eagle's basal medium containing the tritium-labeled compound before the lenses were sectioned or flat mounted. Results obtained with the human lenses were like those obtained with the rat lenses. These studies on the rat lenses and the human senile cataract lenses indicated that the lens

11 490 Hanna Investigative Ophthalmology August 1965 epithelial cells underwent a similar metabolic process. Several "antimetabolites" of nucleic acid intermediates were studied in the rat lenses after anterior chamber injections. Half of the lenses were flat mounted and half were sectioned. The following results were obtained: 3 H-IDU was incorporated into DNA, this labeled DNA appeared in the mitotic figures, and these cells migrated to the nuclear bow region. Cytosine 3 H- arabinoside gave a mixed DNA and RNA label like that obtained with 3 H-cytidine while 5-3 H-fluorouracil gave only an RNA label. Tritium-labeled 6-azauridine gave no labeled cells in the eye. Iodine-131 has been noted by Wheeler, Harding, and Hughes 25 to be incorporated into the germinative zone of cells after lens wounding after in vitro incubation with 131 I-IDU medium. In repeating this experiment with 3 H-IDU in place of the 131 I-IDU, the same results were obtained. This established that the IDU was directly incorporated into DNA. Summary Cell division in the developing mouse lens was studied by thymidine-tritium and autoradiographic techniques. From the time the secondary lens fibers began to form just before birth until the eyes opened at about 12 days, most of the epithelial cells were undergoing almost continuous cell division. The cell division cycle was estimated to be 56 hours during this period. Mouse eye development at the time the lids open corresponded in development to that of a human eye at birth. The number of epithelial cells dividing over the lens decreased markedly after the eyes opened. Evidence was presented that indicated some of the cells in the central area of the lens rested in the G 2 phase of cell division. Cell migration in the young rat lens from the germinative zone to the point where nonnucleated lens fibers formed took about two to four months. It would take an estimated one-half to one year for lens epithelial cells to migrate this distance in the adult rat lens. Cell division in the chicken lens was found in a narrow zone just anterior to the annular pad. These epithelial cells migrated through the annular pad at a slow rate with an estimated transient time of one and one-half years. Ribonucleic acid (RNA) and protein metabolism in the developing mouse lens was studied with the use of uridine-tritium and leucine-tritium, respectively. Nucleated cells undergoing RNA and protein metabolism were found throughout the lens of the newborn mouse and this continued until about 6 days of age. Lens fiber cells in the process of losing their cell nuclei were not undergoing RNA or protein synthesis except in the very young animals through 6 days of age. This was most easily observed in the young chicken lens where the cortex fibers differentiating into nuclear fibers involved a number of cells which do not undergo RNA and protein synthesis. With increasing age of the mouse the uridine-tritium or leucine-tritium did not readily penetrate deep into the lens cortex. In the adult lens of various species, including the senile cataract lens of the human, uridine-tritium and leucine-tritium were found in epithelial cells and in developing fiber cells at the equator region. Tritium from leucine-tritium but not from uridine-tritium was found in the lens capsule. The help of the following people in various aspects of this project is gratefully acknowledged: Travis W. Jenkins, Henry C. Keatts, John E. Slayden, and Katherine P. Wilkinson of the Department of Pharmacology, University of Arkansas Medical Center. REFERENCES 1. Harding, C. V., Hughes, W. L., Bond, V. P., and Schork, P.: Autoradiographic localization of tritiated thymidine in whole mount preparations of lens epithelium, Arch. Ophth. 63: 58, Harding, C. V., Donn, A., and Srinivasan, B. D.: Incorporation of thymidine by injured

12 Volume 4 Number 4 Changes in DNA, RNA, and protein synthesis 491 lens epithelium, Exper. Cell Res. 18: 582, Rothstein, H., and Harding, C. V.: Injuryinduced synthesis of deoxyribonucleic acid in the lens of the sea bass, Nature 194: 294, Harding, C. V., Rothstein, H., and Newman, M. B.: The activation of DNA synthesis and cell division in rabbit lens in vitro, Exper. Eye Res. 1: 457, Harding, C. V., and Thayer, M. N.: DNA synthesis and cell division in the cultured ocular lens, INVEST. OPHTH. 3: 302, Hanna, C, and O'Brien, J. E.: Studies on galactose cataract formation utilizing thymidine-tritium, Arch. Ophth. 64: 708, Hanna., C, and O'Brien, J. E.: Cell production and migration in the epithelial layer of the lens, Arch. Ophth. 66: 103, Brolin, S. E., Diderholmh, H., and Hammar, H.: An autoradiographic study on cell migration in the eye lens epithelium, Acta Soc. med. upsal. 66: 43, Hanna, C, Bicknell, D. S., and O'Brien, J. E.: Cell turnover in the adult eye, Arch. Ophth. 65: 695, Beitalanfly, F. D.: Tritiated thymidine versus colchicine techniques in the study of cell population cytodynamics, Lab. Invest. 13: 871, Thomson, D. S., Pirie, A., and Overall, M.: Autoradiography of lens epithelium after parenteral injection of tritiated thymidine, Arch. Ophth. 67: 464, Mikulicich, A. C, and Young, R. W.: Cell proliferation and displacement in the lens epithelium of young rats injected with tritiated thymidine, INVEST. OPHTH. 2: 344, Scullica, L., Grimes, P., and McElvain, N.: DNA synthesis in the rat lens epithelium after roentgen irradiation, Arch. Ophth. 68: 792, Scullica, L., Grimes, P., and McElvain, N.: Further autoradiographic studies of the lens epithelium normal and x-irradiated, Arch. Ophth. 70: 659, Hanna, C, and O'Brien, J. E.: Lens epithelial cell proliferation and migration in radiation cataracts, Radiation Res. 19: 1, Hanna, C, and O'Brien, J. E.: Effect of AET on x-ray radiation cataracts, Arch, internat. pharmacodyn. 142: 198, Hanna, C, and Wilkinson, K. P.: Uptake of tritium-labeled thymidine in the rabbit cornea infected with herpes simplex, Exper. Eye Res. 3: 36, Gelfant, S.: Initiation of mitosis in relation to the cell division, Exper. Cell Res. 26: 395, Otis, E., and Brent, R.: Equivalent ages in mouse and human embryos, Anat. Rec. 120: 33, Harding, C. V., Feldherr, C, and Srinivasan, B. D.: In Smelser, G. K., editor: The structure of the eye, New York, 1961, Academic Press, Inc., p Pirie, A.: The effect of x-irradiation on the lens of the embryo and the adult hen, Radiation Res. 11: 260, Pirie, A.: Difference in reaction to x-irradiation between chicken and rabbit lens, Radiation Res. 15: 211, Benedict, W. H.: Development of x-ray induced lamellar cataract in the newborn mouse in relation to age at time of irradiation, Tr. Am. Ophth. Soc. 60: 373, Hassan, M., and Greenberg, D. M.: Distribution of label from metabolism of radioactive leucine, norleucine and norvaline in tissue, excreta and respiratory carbon dioxide, Arch. Biochem. & Biophys. 39: 129, Wheeler, M. B., Harding, C. V., and Hughes, W. L.: Incorporation of idoxuridine (IDU) by the rabbit lens epithelium in vitro, Arch. Ophth. 71: 861, 1964.