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Fine structure of lentoid bodies derived from normal and cataractous mouse lenses
Exp. Eye Res. (1980) 31, 535-541
Fine Structure of Lentoid Bodies Derived from Normal and Cataraetous Mouse Lenses F U J I K O L. I-IuANO, I)AUL R U S S E L L AND TOICHIRO KUWABAI~A
Laboratory of Vision Research, National Eye Institute, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, Md 20205, U.S.A. (Received 1 August 1980, New York) Lentoid bodies, aggregations of epithelial calls originating in cultures of lenses from normal and Nakano eataractous strain mice were studied by transmission and scanning electron microscopy. Cells of the lenteid body of the mouse lens epithelium tend to form random aggregations with marked degeneration in the central zone in contrast to the reported structure of the lentoid bodies of the chick. Although the appearances of cells constituting lentoid bodies of both normal and eataractous mice were similar, electron microscopic histochemistry revealed that there was a marked difference in the ATPase activity. The precipitate produced by the histoehemical reaction where phosphate is released by the action of ATPase was abundantly present in the vicinity of cell membranes of lentoid body cells of the normal mouse, whereas it was absent in cells of Nakano strain mice. This is another demonstration that an ATPase inhibitor which is present in the lens of Nakano strain was carried into the cultured cells. Key words: lentoid body; cataract; ATPase; mouse.
1. Introduction The lentoid body, a spherical aggregation of lens epithelial cells in tissue culture, was first reported by Okada, Eguehi and Takeiehi (1971). They described the cells of the lentoid body derived from newly hatched chick lens epithelium as having a cytological appearance similar to that of the mature lens cells and containing delta erystallin lens protein. They concluded that the lentoid bodies did indeed resemble miniature lenses formed in tissue culture. Recently Russell, Fukui, Tsunematsu, tIuang and Kinoshita (1977) produce d similar lentoid bodies from epithelial cells of normal and cataraetous lenses of mammals. The study revealed that the lentoid body which developed in congenital cataractous strain (Nakano) mice contained an adenosine triphosphatase (ATPase) inhibitor which had been demonstrated earlier in whole cataractous lens (Iwata and Kinoshita, 1971). The purpose of this paper is to describe the morphological details of the lentoid body and the electron-microscopic histoehemistry of Na-K ATPase of these cells. 2. Materials and Methods Six to 10 littermates of 4- to 7-week-old mice of normal albino N:GP(S) and cataractous Nakano strain mice were used for establishment of culture lines. The lens epithelium was excised together with the capsule from freshly enucleated eyes. After a brief washing in a solution of 0.05o/0 EDTA, the tissue was minced and small explants were placed on culture plates. The culture was carried out under 5 % CO 2 and 95 % air at 37~ and the medium consisting of RPMI1640 and 10~ fetal calf serum (GIBCO), was changed every 2 days. After about 2 weeks, the cells were subcultured every 5 days. Methods for determination of :/-crystallin content and other biochemical studies have been described in detail in a previous report (Russell, Fukui, Tsunematsu, Huang and Kinoshita, 1977). Reprint requests to: Toiehiro Kuwabara, Laboratory of Vision Research, NEI, 9000 Rockville Pike, Bldg.(}, Room 213, Bethesda, Md 20205, U.S.A. (~ 1980 Academic Press Inc. (London) Limited
0014-4835/80/110535+07 $01.00/0 535
536
F.L. HUANG, P. RUSSELL AND T. KUWABAI~A
For cytological studies, the growing cells were fixed in toto with a 4~o glutaraldehyde solution in 0-15 M-phosphate buffer (pH 7-2) at room temperature. After a 30 min fixation, the plates were postfixed for 90 min in 1% osmium tetroxide at 4~ The fixed plates were dehydrated with ethyl alcohol and embedded in an epoxy resin in toto. Propylene oxide treatment was omitted when plastic culture plates were used. The embedded cells and lentoid bodies were examined under a microscope and desired areas of the plate were dissected out using a fine saw. Sections cut at 0.5~m were stained with toluidine blue and examined by light microscopy: Ultrathin sections stained with uranyl acetate and lead citrate were examined by transmission electron microscopy. The lens epithelial cells and lentoid bodies which had been cultured on small pieces of cover slips were examined by scanning electron microscopy. The cultured cells on the glass were; thoroughly washed with a physiological saline solution before fixation in 4 % glutaraldehyde for 30 rain. Then the cover slips were transferred into a mixture of equal amounts o f 4 % glutaraldehyde and 10% neutral formalin. The specimens were kept in this solution for a few days: The pieces of cover slips were thoroughly washed and dehydrated in a series of ethyl alcohol and by a critical point drying technique. The dried samples were mounted on metal specimen holders~ coated with gold-palladium and examined by scanning electron microscopy using an accelerating voltage of 15 kV. ATPase was demonstrated on the lentoid body by the Wachstein-Meisel technique (Wachstein and Meisel, 1957) modified for electron microscopy by Palva and Palkama (1976). The culture plates were briefly fixed in a 2.5% glutaraldehyde solution in Tris buffer pH 7.2 for 5 min at 4~ followed by a thorough washing with the same buffer solution. The plate was incubated in a medium composed of 3 mM-ATP, 2 m~-Pb(N08)~, 3 m~-Mg-SO4, 70 m~-NaC1, 70 m~-KC1 and 0-2 M-TrisHC1 buffer. The pH of the incubation medium was adjusted at 7-2 and the incubation lasted for 30 min at 37~ The samples were washed in 0.33 x-sucrose and postfixed in one percent osmium tetroxide, prepared in Tris buffer, and processed for epoxy embedding for electron microscopy. As a control, pieces of the culture plate were incubated in the same incubation medium omitting ATP and/or adding 0.3 mM-ouabain.
3. Results Following a rest period of a few days, the explanted lens epithelial cells of both normal and cataraetous lenses spread out as a sparsely distributed single layer of flat cells. The doubling time o f subcultured cells was about 31 hr. Subcultured flat epithelial cells extended a few stellar processes about 70-150#m long. Cells were loosely attached to each other mainly by their processes, and partially overlapped, but a solid mosaic sheet was not formed. The surface of these cells was generally smooth and possessed a small number of short microvilli. The profile of the flat oval nucleus was visible on the cell surface. Tips of the processes were finely branched into a brush pattern. Transmission electron microscopy revealed that the cytoplasm, measuring about 2-5 # m in thickness was eleetrolucent and contained a moderate amount of microfilaments. The number Of microorganelles was relatively small. Mitochondria were small and the matrix was somewhat electron dense. A thin layer of basal lamina was present at the basal surface.
Lentoid body of normal albino mice After growing for about 2 weeks the flat epithelial cells began to form aggregations. Without any noticeable prelude, a portion of a flat cell suddenly retracted the cytoplasm and formed a bump. The whole cell eventually became spindled or spherical in shape and started to proliferate (Fig. 1 A, B). This phenomenon occurred randomly in many cells at various times. During the formation of lentoid bodies a few round highly reflectile bodies, measuring about 10/~m in diameter, protruded from the cell mass. These were probably overgrown or degenerating cells. These islands of epithelial cells became spherical mounds measuring 100-200/zm in diameter and often 200/~m in height within a few weeks (Fig. 2). The ability to form lentoid bodies was retained in all cell lines subcultured for over one year.
LENTOID
BODY
537
FIG. 1, (A) Lentoid body of the normal mouse cell at its early stage. Aggregation of cells begins at a portion of an epithelial cell Which has retracted fiat cytoplasm, Scanning electron microscopy ( • 1000). (B) Cross-section of an early lentoid body. Cells are loosely attached to each other, Abundant basal lamina substance (arrows) is p r e s e n t b e n e a t h the cell ( • 6000).
FIG. 2. A well-grown lentoid body of a normal mouse lens measuring about 100#m in diameter and 200 # m in height. Scanning electron microscopy ( x 500).
II
EER
3][
538
F . L . H U A N G , P. R U S S E L L
A N D T. K U W A B A R A
FIo. 3. Cross-sectional view of a relatively fiat lentoid body. The aggregated cells abruptly form a mound. The central portion is degenerative. Toluidine blue staining ( • 800; inset • 22).
Fie. 4. (A) Superficial zone of a large lentoid body derived from Nakano strain mouse. Cells are generally spindle in shape and closely attached. Toluidine blue staining ( • 800). (B) Cells in the superficial zone of the lentoid body shown above. Cells show well-developed junctions (arrows) and a moderate lentieular differentiation- sparse microorganelles and dense fine granular substances in the cytoplasm ( • 9700).
L E N T O I D BODY
539
The transition from flat cells to lentoid body was abrupt. The lentoid body consisted of looseiy packed cells in the periphery and degenerating cells in the center (Fig. 3). Cells were generally polygonal in shape and had numerous processes. However , the cells in the superficial zone were elongated and relatively closely packed, a n d attached to each other with gap junctions (Fig. 4): The surfaces of these cells were smooth. The cytoplasm contained a moderate number of microorganelles in the finely granular matrix. The appearance of the granules was identical to that of the lens protein. Some lentoid bodies contained numerous lamellar inclusion bodies and various particles. Varying amounts of basal lamina were formed in the intercellular spaces. Cells of the superficial layer of older lentoid bodies were .more compactly packed and their basal lamina substance was abundant.
Fro. 5. (A) Lead particles, a product of Na~-K ATPase activity, are present along the cell membrane of a lentoid body of a normal mouse. Wachstein-Meisel incubation ( • 7200). (:B) The lead particles are markedly sparse in a lentoid body derived from Nakano cataractous lens epithelium. WachsteinMeisel incubation ( • 7200). Cells in the central zone of the lentoid body were markedly degenerated, but their original cell s~ape did not disintegrate and was relatively well maintained. Unlike ordinary karyolysis, some nuclei of these cells often showed prominent nucleoli and swollen nuclear envelopes. Lead precipitations, an indication of the presence of N a - K ATPase activity, were uniformly formed along the cell membranes of the cells in the superficial zone (Fig. 5A). The degenerating cells were generally negative in enzyme activity. However, small aggregations of the lead precipitate were occasionally present within the cytoplasm. The enzyme activity was almost completely inhibited by addition of 0.3 mM-ouabain in the incubation medium, and the reaction was negative following incubation of the tissue without ATP in the medium.
Lentoid body of Nakano mouse The epithelial cell obtained from Nakano strain cataraetous lenses grew in the tissue culture system similarly to the normal mouse lens. However, the clone-forming ability II-2
540
F. L. H U A N G ,
P. R U S S E L L
A N D T. K U W A B A l ~ A
was about one half of that of the normal lens epithelium. The electron microscopic appearance of the cells of the mono-layer culture and of the lentoid body did not differ from that of the normal mouse cell. The striking difference in the lentoid body which was derived from Nakano strain was a marked reduction of ATPase activity. The lead precipitation was absent in the cells of the superficial zone and was only sparsely distributed along degenerating cells (Fig. 5B). The enzyme reaction was completely inhibited following incubation with ouabain.
4. Discussion As previously described, lens epithelial cells of normal and cataractous mice have the capability to form lentoid bodies following tissue culture (Russell et al., 1977). However, the appearance of cells in this experiment differs somewhat from that of the lentoid body derived from embryonal chick lenses by Okada and his associates (Okada, Eguehi and Takeichi, 1971). Although a moderate maturation-sparse microorganelles and production of 7-erystallin- was demonstrated, the cells of mouse lentoid bodies have not differentiated as highly as the chick cell. Cells in the chick lentoid body are described as sh owing a cytologic appearance indistinguishable from matured lens fibers. They are firmly attached to each other and the cytoplasm contains only the granular lens protein substance. Also, the ceils form an onion-like regular arrangement in which each cell has a basal attachment to the culture plate. The lentoid body of mouse cells in the present study consists mainly of loosely aggregated polygonal cells in the periphery and degenerating cells in the center. Besides the species difference, which may be the most undeniable factor, there are some differences in culture methods used by Okada's and in the present study. The mouse lens epithelium was cultured for over a year with many subcultures, whereas Okada's lentoid body was produced mainly in the primary culture. Repeated use of trypsin may result in alterations of cell surfaces and cytoplasmic characteristics. The aggregates of the present mouse lentoid bodies are so large that diffusion of essential nutrients into the central area would be slow. This may be another reason for the extensive degeneration in the lentoid body of this study. Nonetheless, despite the difference in degree of differentiation in both studies, lens cells are shown to form lentoid bodies which maintain a maturation capability. Electron-microscopic histoehemistry has clearly demonstrated the locational distribution of ATPase activity in the lentoid body of the mouse. The lead particles which indicate enzyme activity are mainly present in association with the cell membrane. This distribution is identical to that of normal lens epithelium (1%lva and Palkama, 1976). The present study has demonstrated the markedly low activity in lentoid bodies of Nakano cataract lens, confirming the results of our previous work suggesting the presence of a N a - K ATPase inhibitor in cultured cells of this strain (Russell et al., 1977). As Iwata and Kinoshita (1971) have proposed, deficiency of N a - K ATPase may be related to cataractogenesis. However, no appreciable difference in the degree of degeneration of the lentoid bodies between normal and cataractous strains has been demonstrated. Central portions of both lentoid bodies are equally degenerative. Since the Nakano epithelial cells in culture as well as the cells in the whole lens remain viable, it may indicate that residual ATPase activity is sufficient for cell growth, or suggest the presence of a compensating metabolic change in these cells. Our findings are the first histological demonstration of reduced N a - K ATPase of cultured cells of the lens epithelium.
L E N T O I D BODY
541
REFERENCES Iwata, S. and Kinoshita, J. H. (1971). Mechanism of development of hereditary cataract in mice. Invest. Ophthalmol. 10, 504-12. Okada, T. S., Eguchi, G. and Takeichi, M. (1971). The expression of dif[erentiation by chicken lens epithelium in vitro cell culture. Dev. Growth Differ. 13, 323-36. Palva, M. and Palkama, A. (1976). Electronmicroscopical, histochemical and biochemical findings on the Na-K-ATPase activity in the epithelium of the rat lens. Exp. Eye ties. 22, 229-36. Russell, P., Fukui, H.N., Tsunematsu, u Huang, F. L. and Kinoshita, J. H. (1977). Tissue culture of lens epithelial cells from normal and Nakano mice. Invest. Ophthalmol. 16, 243-6. Wachstein, M. and Meisel, E. (1957). Histoehemistry of hepatic phosphatases at a physiological pFI. Am. J. Clin. Path. 27, 13-23.
Fine Structure of Lentoid Bodies Derived from Normal and Cataraetous Mouse Lenses F U J I K O L. I-IuANO, I)AUL R U S S E L L AND TOICHIRO KUWABAI~A
Laboratory of Vision Research, National Eye Institute, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, Md 20205, U.S.A. (Received 1 August 1980, New York) Lentoid bodies, aggregations of epithelial calls originating in cultures of lenses from normal and Nakano eataractous strain mice were studied by transmission and scanning electron microscopy. Cells of the lenteid body of the mouse lens epithelium tend to form random aggregations with marked degeneration in the central zone in contrast to the reported structure of the lentoid bodies of the chick. Although the appearances of cells constituting lentoid bodies of both normal and eataractous mice were similar, electron microscopic histochemistry revealed that there was a marked difference in the ATPase activity. The precipitate produced by the histoehemical reaction where phosphate is released by the action of ATPase was abundantly present in the vicinity of cell membranes of lentoid body cells of the normal mouse, whereas it was absent in cells of Nakano strain mice. This is another demonstration that an ATPase inhibitor which is present in the lens of Nakano strain was carried into the cultured cells. Key words: lentoid body; cataract; ATPase; mouse.
1. Introduction The lentoid body, a spherical aggregation of lens epithelial cells in tissue culture, was first reported by Okada, Eguehi and Takeiehi (1971). They described the cells of the lentoid body derived from newly hatched chick lens epithelium as having a cytological appearance similar to that of the mature lens cells and containing delta erystallin lens protein. They concluded that the lentoid bodies did indeed resemble miniature lenses formed in tissue culture. Recently Russell, Fukui, Tsunematsu, tIuang and Kinoshita (1977) produce d similar lentoid bodies from epithelial cells of normal and cataraetous lenses of mammals. The study revealed that the lentoid body which developed in congenital cataractous strain (Nakano) mice contained an adenosine triphosphatase (ATPase) inhibitor which had been demonstrated earlier in whole cataractous lens (Iwata and Kinoshita, 1971). The purpose of this paper is to describe the morphological details of the lentoid body and the electron-microscopic histoehemistry of Na-K ATPase of these cells. 2. Materials and Methods Six to 10 littermates of 4- to 7-week-old mice of normal albino N:GP(S) and cataractous Nakano strain mice were used for establishment of culture lines. The lens epithelium was excised together with the capsule from freshly enucleated eyes. After a brief washing in a solution of 0.05o/0 EDTA, the tissue was minced and small explants were placed on culture plates. The culture was carried out under 5 % CO 2 and 95 % air at 37~ and the medium consisting of RPMI1640 and 10~ fetal calf serum (GIBCO), was changed every 2 days. After about 2 weeks, the cells were subcultured every 5 days. Methods for determination of :/-crystallin content and other biochemical studies have been described in detail in a previous report (Russell, Fukui, Tsunematsu, Huang and Kinoshita, 1977). Reprint requests to: Toiehiro Kuwabara, Laboratory of Vision Research, NEI, 9000 Rockville Pike, Bldg.(}, Room 213, Bethesda, Md 20205, U.S.A. (~ 1980 Academic Press Inc. (London) Limited
0014-4835/80/110535+07 $01.00/0 535
536
F.L. HUANG, P. RUSSELL AND T. KUWABAI~A
For cytological studies, the growing cells were fixed in toto with a 4~o glutaraldehyde solution in 0-15 M-phosphate buffer (pH 7-2) at room temperature. After a 30 min fixation, the plates were postfixed for 90 min in 1% osmium tetroxide at 4~ The fixed plates were dehydrated with ethyl alcohol and embedded in an epoxy resin in toto. Propylene oxide treatment was omitted when plastic culture plates were used. The embedded cells and lentoid bodies were examined under a microscope and desired areas of the plate were dissected out using a fine saw. Sections cut at 0.5~m were stained with toluidine blue and examined by light microscopy: Ultrathin sections stained with uranyl acetate and lead citrate were examined by transmission electron microscopy. The lens epithelial cells and lentoid bodies which had been cultured on small pieces of cover slips were examined by scanning electron microscopy. The cultured cells on the glass were; thoroughly washed with a physiological saline solution before fixation in 4 % glutaraldehyde for 30 rain. Then the cover slips were transferred into a mixture of equal amounts o f 4 % glutaraldehyde and 10% neutral formalin. The specimens were kept in this solution for a few days: The pieces of cover slips were thoroughly washed and dehydrated in a series of ethyl alcohol and by a critical point drying technique. The dried samples were mounted on metal specimen holders~ coated with gold-palladium and examined by scanning electron microscopy using an accelerating voltage of 15 kV. ATPase was demonstrated on the lentoid body by the Wachstein-Meisel technique (Wachstein and Meisel, 1957) modified for electron microscopy by Palva and Palkama (1976). The culture plates were briefly fixed in a 2.5% glutaraldehyde solution in Tris buffer pH 7.2 for 5 min at 4~ followed by a thorough washing with the same buffer solution. The plate was incubated in a medium composed of 3 mM-ATP, 2 m~-Pb(N08)~, 3 m~-Mg-SO4, 70 m~-NaC1, 70 m~-KC1 and 0-2 M-TrisHC1 buffer. The pH of the incubation medium was adjusted at 7-2 and the incubation lasted for 30 min at 37~ The samples were washed in 0.33 x-sucrose and postfixed in one percent osmium tetroxide, prepared in Tris buffer, and processed for epoxy embedding for electron microscopy. As a control, pieces of the culture plate were incubated in the same incubation medium omitting ATP and/or adding 0.3 mM-ouabain.
3. Results Following a rest period of a few days, the explanted lens epithelial cells of both normal and cataraetous lenses spread out as a sparsely distributed single layer of flat cells. The doubling time o f subcultured cells was about 31 hr. Subcultured flat epithelial cells extended a few stellar processes about 70-150#m long. Cells were loosely attached to each other mainly by their processes, and partially overlapped, but a solid mosaic sheet was not formed. The surface of these cells was generally smooth and possessed a small number of short microvilli. The profile of the flat oval nucleus was visible on the cell surface. Tips of the processes were finely branched into a brush pattern. Transmission electron microscopy revealed that the cytoplasm, measuring about 2-5 # m in thickness was eleetrolucent and contained a moderate amount of microfilaments. The number Of microorganelles was relatively small. Mitochondria were small and the matrix was somewhat electron dense. A thin layer of basal lamina was present at the basal surface.
Lentoid body of normal albino mice After growing for about 2 weeks the flat epithelial cells began to form aggregations. Without any noticeable prelude, a portion of a flat cell suddenly retracted the cytoplasm and formed a bump. The whole cell eventually became spindled or spherical in shape and started to proliferate (Fig. 1 A, B). This phenomenon occurred randomly in many cells at various times. During the formation of lentoid bodies a few round highly reflectile bodies, measuring about 10/~m in diameter, protruded from the cell mass. These were probably overgrown or degenerating cells. These islands of epithelial cells became spherical mounds measuring 100-200/zm in diameter and often 200/~m in height within a few weeks (Fig. 2). The ability to form lentoid bodies was retained in all cell lines subcultured for over one year.
LENTOID
BODY
537
FIG. 1, (A) Lentoid body of the normal mouse cell at its early stage. Aggregation of cells begins at a portion of an epithelial cell Which has retracted fiat cytoplasm, Scanning electron microscopy ( • 1000). (B) Cross-section of an early lentoid body. Cells are loosely attached to each other, Abundant basal lamina substance (arrows) is p r e s e n t b e n e a t h the cell ( • 6000).
FIG. 2. A well-grown lentoid body of a normal mouse lens measuring about 100#m in diameter and 200 # m in height. Scanning electron microscopy ( x 500).
II
EER
3][
538
F . L . H U A N G , P. R U S S E L L
A N D T. K U W A B A R A
FIo. 3. Cross-sectional view of a relatively fiat lentoid body. The aggregated cells abruptly form a mound. The central portion is degenerative. Toluidine blue staining ( • 800; inset • 22).
Fie. 4. (A) Superficial zone of a large lentoid body derived from Nakano strain mouse. Cells are generally spindle in shape and closely attached. Toluidine blue staining ( • 800). (B) Cells in the superficial zone of the lentoid body shown above. Cells show well-developed junctions (arrows) and a moderate lentieular differentiation- sparse microorganelles and dense fine granular substances in the cytoplasm ( • 9700).
L E N T O I D BODY
539
The transition from flat cells to lentoid body was abrupt. The lentoid body consisted of looseiy packed cells in the periphery and degenerating cells in the center (Fig. 3). Cells were generally polygonal in shape and had numerous processes. However , the cells in the superficial zone were elongated and relatively closely packed, a n d attached to each other with gap junctions (Fig. 4): The surfaces of these cells were smooth. The cytoplasm contained a moderate number of microorganelles in the finely granular matrix. The appearance of the granules was identical to that of the lens protein. Some lentoid bodies contained numerous lamellar inclusion bodies and various particles. Varying amounts of basal lamina were formed in the intercellular spaces. Cells of the superficial layer of older lentoid bodies were .more compactly packed and their basal lamina substance was abundant.
Fro. 5. (A) Lead particles, a product of Na~-K ATPase activity, are present along the cell membrane of a lentoid body of a normal mouse. Wachstein-Meisel incubation ( • 7200). (:B) The lead particles are markedly sparse in a lentoid body derived from Nakano cataractous lens epithelium. WachsteinMeisel incubation ( • 7200). Cells in the central zone of the lentoid body were markedly degenerated, but their original cell s~ape did not disintegrate and was relatively well maintained. Unlike ordinary karyolysis, some nuclei of these cells often showed prominent nucleoli and swollen nuclear envelopes. Lead precipitations, an indication of the presence of N a - K ATPase activity, were uniformly formed along the cell membranes of the cells in the superficial zone (Fig. 5A). The degenerating cells were generally negative in enzyme activity. However, small aggregations of the lead precipitate were occasionally present within the cytoplasm. The enzyme activity was almost completely inhibited by addition of 0.3 mM-ouabain in the incubation medium, and the reaction was negative following incubation of the tissue without ATP in the medium.
Lentoid body of Nakano mouse The epithelial cell obtained from Nakano strain cataraetous lenses grew in the tissue culture system similarly to the normal mouse lens. However, the clone-forming ability II-2
540
F. L. H U A N G ,
P. R U S S E L L
A N D T. K U W A B A l ~ A
was about one half of that of the normal lens epithelium. The electron microscopic appearance of the cells of the mono-layer culture and of the lentoid body did not differ from that of the normal mouse cell. The striking difference in the lentoid body which was derived from Nakano strain was a marked reduction of ATPase activity. The lead precipitation was absent in the cells of the superficial zone and was only sparsely distributed along degenerating cells (Fig. 5B). The enzyme reaction was completely inhibited following incubation with ouabain.
4. Discussion As previously described, lens epithelial cells of normal and cataractous mice have the capability to form lentoid bodies following tissue culture (Russell et al., 1977). However, the appearance of cells in this experiment differs somewhat from that of the lentoid body derived from embryonal chick lenses by Okada and his associates (Okada, Eguehi and Takeichi, 1971). Although a moderate maturation-sparse microorganelles and production of 7-erystallin- was demonstrated, the cells of mouse lentoid bodies have not differentiated as highly as the chick cell. Cells in the chick lentoid body are described as sh owing a cytologic appearance indistinguishable from matured lens fibers. They are firmly attached to each other and the cytoplasm contains only the granular lens protein substance. Also, the ceils form an onion-like regular arrangement in which each cell has a basal attachment to the culture plate. The lentoid body of mouse cells in the present study consists mainly of loosely aggregated polygonal cells in the periphery and degenerating cells in the center. Besides the species difference, which may be the most undeniable factor, there are some differences in culture methods used by Okada's and in the present study. The mouse lens epithelium was cultured for over a year with many subcultures, whereas Okada's lentoid body was produced mainly in the primary culture. Repeated use of trypsin may result in alterations of cell surfaces and cytoplasmic characteristics. The aggregates of the present mouse lentoid bodies are so large that diffusion of essential nutrients into the central area would be slow. This may be another reason for the extensive degeneration in the lentoid body of this study. Nonetheless, despite the difference in degree of differentiation in both studies, lens cells are shown to form lentoid bodies which maintain a maturation capability. Electron-microscopic histoehemistry has clearly demonstrated the locational distribution of ATPase activity in the lentoid body of the mouse. The lead particles which indicate enzyme activity are mainly present in association with the cell membrane. This distribution is identical to that of normal lens epithelium (1%lva and Palkama, 1976). The present study has demonstrated the markedly low activity in lentoid bodies of Nakano cataract lens, confirming the results of our previous work suggesting the presence of a N a - K ATPase inhibitor in cultured cells of this strain (Russell et al., 1977). As Iwata and Kinoshita (1971) have proposed, deficiency of N a - K ATPase may be related to cataractogenesis. However, no appreciable difference in the degree of degeneration of the lentoid bodies between normal and cataractous strains has been demonstrated. Central portions of both lentoid bodies are equally degenerative. Since the Nakano epithelial cells in culture as well as the cells in the whole lens remain viable, it may indicate that residual ATPase activity is sufficient for cell growth, or suggest the presence of a compensating metabolic change in these cells. Our findings are the first histological demonstration of reduced N a - K ATPase of cultured cells of the lens epithelium.
L E N T O I D BODY
541
REFERENCES Iwata, S. and Kinoshita, J. H. (1971). Mechanism of development of hereditary cataract in mice. Invest. Ophthalmol. 10, 504-12. Okada, T. S., Eguchi, G. and Takeichi, M. (1971). The expression of dif[erentiation by chicken lens epithelium in vitro cell culture. Dev. Growth Differ. 13, 323-36. Palva, M. and Palkama, A. (1976). Electronmicroscopical, histochemical and biochemical findings on the Na-K-ATPase activity in the epithelium of the rat lens. Exp. Eye ties. 22, 229-36. Russell, P., Fukui, H.N., Tsunematsu, u Huang, F. L. and Kinoshita, J. H. (1977). Tissue culture of lens epithelial cells from normal and Nakano mice. Invest. Ophthalmol. 16, 243-6. Wachstein, M. and Meisel, E. (1957). Histoehemistry of hepatic phosphatases at a physiological pFI. Am. J. Clin. Path. 27, 13-23.