|Molecular Vision 2004;
Received 11 December 2003 | Accepted 28 January 2004 | Published 2 February 2004
DNase I and fragmented chromatin during nuclear degradation in adult bovine lens fibers
Alicia De María,
Laboratorio de Cultivo de Tejidos, Sección Biología Celular, Departamento de Biología Celular y Molecular, Facultad de Ciencias, Universidad de la República, Montevideo, Uruguay
Correspondence to: Cristina Arruti, Laboratorio de Cultivo de Tejidos, Sección Biología Celular, Facultad de Ciencias, Iguá 4225, 11400 Montevideo, Uruguay; FAX: (5982) 525 86 29; email: firstname.lastname@example.org
Purpose: Nuclear loss is a most remarkable organelle disappearance during terminal differentiation of lens fiber cells given that it implicates the full degradation of a major molecular component, DNA. Consequently, to gain insight into the progression of DNA cleavage we analyzed the appearance of single strand breaks in relationship with chromatin condensation. To assess a possible involvement of DNase I in DNA fragmentation we explored its localization in lens fibers having different degrees of nuclear breakdown, evaluated by the state of chromatin, nuclear envelope, and DNA.
Methods: Whole mounts of adult bovine lens epithelium as well as lens cryosections were utilized to examine, using antibodies or specific molecular probes, the localization of DNase I, nuclear membrane, lamins, and DNA 3'-OH-free termini. Nuclease activity gel and western blot assays were used to characterize DNase I in different lens fiber extracts.
Results: Nuclear morphology was found to undergo significant changes from the onset of fiber differentiation. Initial spherical nuclei present at early fibergenesis stages evolve to elongated ones in mature fibers. Chromatin did not present signs of condensation in these nuclei. However, nuclei from fibers located deeper in lens volume exhibited some chromatin condensation and fragmentation while the nuclear lamina appeared undamaged. At more advanced stages, different patterns of nuclear envelope integrity and chromatin condensation and cleavage were observed. DNase I was found in the cytoplasm in the very initial fibers and then in the nuclear territory. DNase I appeared closely associated with fully condensed and fragmented chromatin at the final phases of nuclear breakdown.
Conclusions: DNase I is a nuclease present in bovine lens fibers and can be considered as an enzyme producing final DNA cleavage since it is closely associated with highly fragmented DNA in disintegrating nuclei.
The ocular lens is a strange organ in which anucleated cells are stable constituents. Furthermore, these anucleated cells form the major part of the organ volume. It is known, from the beginning of research on vertebrate lens anatomy, that these cells lose their nuclei during a terminal differentiation process. Many years ago, a milestone article by Appleby and Modak  communicated that DNA is cleaved, during nuclear loss, by a degradation mechanism generating fragments of defined sizes. Fragments that produce ladders once separated by electrophoresis in agarose gels. Since that time many efforts have been devoted to elucidate the properties of this DNA degradation. One of the most important concerns has been the identification of the nuclease. Up to now different putative nucleases have been found in lenses. DNase I, L-DNase II and the caspase dependent nuclease (DFF) have been identified in chick lenses [2-4]. More recently, DLAD was identified as responsible for DNA degradation in mouse lens . It is interesting to note that neither DNase I nor DFF have been found in mouse lens [6,7]. However, notwithstanding the possible existence of differences amongst species, it is conceivable that more than one nuclease could be involved in complete lens fiber DNA fragmentation.
DNA degradation in secondary lens fiber cells has a unique property when compared to DNA cleavage processes occurring in other cells. Fragmented DNA does not leave the cell. For instance, in the majority of cell types nuclear remnants resulting from apoptosis are extruded to the extra cellular space as a constituent of apoptotic bodies. Then they are either engulfed by other cells or directly eliminated to a body cavity . In lens fiber cells complete DNA catabolism is achieved inside the cell itself. Bearing in mind this fact, we recently hypothesized that a nuclease, having the catalytic properties of DNase I, could be a putative nuclease participating in lens cell DNA degradation.
We have recently described the organization of the bovine DNase I gene, its expression as well as the sub cellular localization of the enzyme in epithelial lens cells . Our results showed that the enzyme is localized in organelles of the secretory pathway and that it appears associated with highly fragmented chromatin at the final stages of apoptosis. Hence, we wondered whether DNase I could be present and involved in some stages of DNA degradation during bovine lens fiber cell terminal differentiation. The results presented here indicate that there is an important translocation of the enzyme during fiber differentiation. Whereas it is mainly cytoplasmic in epithelial cells, a noticeable fraction of the enzyme appears associated to the nucleus of mature fiber cells. Actually, at the onset of nuclear breakdown DNase I is at the nuclear periphery but then becomes closely associated with highly fragmented chromatin remnants at the final stages of nuclear disintegration.
Adult bovine eyes (3-4 years old) were obtained from a local abattoir (Frigorifíco Matadero Carrasco, Montevideo, Uruguay) and processed within 4 h after slaughter. Lenses were immediately used to prepare protein samples, to make whole mounts as described  or were frozen in liquid nitrogen for cryosection.
Two polyclonal rabbit anti-bovine DNase I antibodies were used; AbR (Rockland, Gilbertsville, PA) and Ab2983 prepared by us . For lamins immunodetection the monoclonal antibody L3f4 was used . A goat anti-rabbit immunoglubulin peroxidase-conjugated antibody (Gibco Invitrogen, Carlsbad, CA) was used for western blotting. For in situ immunodetection, rabbit primary antibodies were detected using FITC (Gibco Invitrogen, Carlsbad, CA) or Alexa 488 (green, Molecular Probes Inc., Eugene, OR) labeled antibodies. Mouse primary antibody was detected using a TRITC (Sigma, St. Louis, MO) labeled secondary antibody.
Detection of DNase I activity
Nuclease activity gel assays were performed in 12% T/0.9% C polyacrylamide gels containing double stranded high molecular weight DNA (30 μg/ml) as described . Gels were incubated in activity buffer (10 mM Tris-HCl pH 7.5, 10 mM CaCl2, 10 mM MgCl2) overnight and finally stained with 1% Toluidine blue.
SDS-PAGE and western blot
SDS-PAGE was performed in 12% T/0.9% C polyacrylamide gels. Proteins were electrotransferred onto nitrocellulose membranes and blocked with 3% (w/v) non-fat milk, 1 h at room temperature (RT) and antibody Ab2983 was incubated overnight at 4 °C. The secondary antibody (1:1500) was incubated 1 h at RT and developed by quimioluminiscence with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL) or ECL kits (Amersham-Pharmacia Biotech, Little Chalfont, UK).
In situ immunodetection
For DNase I immunodetection lens whole mounts and cryosections were fixed in 4% (w/v) paraformaldehyde (PAF) and permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MO) prior to blocking with 3% (w/v) bovine serum albumin (BSA). Anti-DNase I antibodies were incubated at 4 °C overnight and the secondary antibody for 1 h at RT. For lamins immunodetection cryosections were fixed at -20 °C in methanol for 10 min followed by acetone for 1 min. After blocking with 3% (w/v) BSA, antibody L3f4 was incubated for 1 h at RT and the secondary antibody for 1 h at RT. In all sections nuclear counterstaining was done with 0.0005% (w/v) Hoechst 33342 (Molecular Probes, Eugene, OR). Labeled whole mounts and sections were examined and photographed using a Microphot FX-A microscope equipped with epifluorescence (Nikon, Japan).
Labeling of membrane-bound organelles
PAF fixed sections were incubated with DiOC6 (1 μg/ml, Molecular Probes, Eugene, OR) for 10 min. Nuclear counterstaining was done with 0.0005% (w/v) Hoechst 33342 (Molecular Probes, Eugene, OR). Labeled sections were observed under the microscope as mentioned above.
Lens cryosections were fixed in 4% (w/v) PAF for 40 min and permeabilized with 0.1% Triton X-100. After blocking with 3% (w/v) BSA, the DNA free 3'-OH ends were labeled using terminal deoxynucleotidyl transferase (TdT) according to manufacturer instructions (Boehringer-Roche, Mannheim, Germany). Biotinilated nucleotides were detected using rhodamine-conjugated streptavidin (Sigma, St. Louis, MO). In some experiments, after the TUNEL assay DNase I immunodetection was performed as described above. For negative controls, TdT was withdrawn from the reaction mix, while for positive controls lens cryosections were pre-incubated with DNase I (15 U/ml) for 5 min. Nuclear counterstaining was done with 0.0005% (w/v) Hoechst 33342 (Molecular Probes, Eugene, OR). Sections were observed under the microscope as mentioned above.
Nuclear breakdown during fiber differentiation
We first directly compared lens nuclear morphology at different stages of lens fiber differentiation. Figure 1 shows nuclei of bovine lens cells at early stages of fibergenesis, using whole mounts of lens capsules with adhered tissue. Lens fiber nuclear shape goes through conspicuous changes during terminal cell differentiation. Bovine secondary lens fibers, as in almost all vertebrate lenses, originate after a final mitosis in cells located at the lens equator. As there is a continuous production of new cells they appear to change location by a "vis a tergo" mechanism, they seem to move toward sub-equatorial lens regions (discussed in ). The differentiating lens fibers elongate and their extremities progress in the direction of the anterior and posterior lens periphery, where they will reach and integrate with lens sutures. During the initial stages of this process fiber nuclei are large, round shaped and display a particularly uncondensed chromatin, as evidenced here by classical staining with ferric hematoxylin (Figure 1A,B). During the main phases of fiber elongation, nuclei also become elongated and have loose chromatin (Figure 1C). These cells reside in slightly deeper regions into the lens cortex. The progression of lens fiber differentiation is marked by an extensive organelle loss (recent review in ). Some time later nuclear morphology changes again. It becomes more oval, then rounded, and begins to exhibit regions of condensed chromatin before fully shrinking. Therefore, fiber nuclei are rounded, then elongate to a maximum and round up once more before they collapse and become nuclear remnants.
To analyze the condensation and fragmentation state of chromatin in these different stages of nuclear degradation, DNA and its free 3'-OH ends were double labeled with Hoechst 33342 and TUNEL using sagital lens cryosections. In the cortex of the lens, where young fibers are localized, DNA staining did not show any sign of chromatin condensation. These nuclei presented an overall morphology ranging from round (outer cortex) to elongated (mid cortex) and their chromatin was not labeled by TUNEL. Figure 2A,G,M show DNA staining and TUNEL assay labeling of one fiber nucleus located at the mid cortex. The first nuclei showing evidences of chromatin condensation were those that had already started to round up. These slightly oval shaped nuclei, found deeper in the lens cortex, presented regions of chromatin condensation, some of them faintly labeled by TUNEL (Figure 2B,H,N). Lens fibers having completely condensed chromatin were found at a deeper level than the previously described cells. Interestingly, we found different chromatin distributions in cells located at the same lens depth, suggesting that they represent alternative nuclear breakdown modalities. Actually, in addition to the known homogeneously condensed nuclei (type A, Figure 2C), some cells exhibited condensed chromatin as a fine, round, peripheral shell-like structure encircling one or two aggregates (type B, Figure 2D), whereas other presented condensed chromatin as isolated, scattered clumps of various sizes (type C, Figure 2E). Chromatin, in type A, type B, or type C patterns, was strongly labeled by TUNEL (Figure 2I,J,K). Moreover, as the merged images indicate, all regions of condensed chromatin were TUNEL positive (Figure 2O,P,Q). The ultimate stages of nuclear disappearance exhibited either a unique or few very small round shaped nuclear remnants, observed as hyper chromatic and TUNEL positive structures (Figure 2F,L,R). No TUNEL labeling was noticed on any nuclear material when the sections were incubated without terminal deoxynucleotidyl transferase (data not shown).
To assess the state of nuclear envelope during the phases of nuclear breakdown described above, the lipophilic probe DiOC6 was used to label the nuclear membrane in the lens sections. DiOC6 labeling showed that the nuclear membrane in the outermost and mid cortical fibers was clearly undamaged (Figure 3A,B). The merge of DiOC6 and Hoechst 33342 staining images showed the membrane surrounding the DNA (Figure 3I,J). Nuclear material with condensation pattern type B exhibited a nuclear membrane which appeared to be undamaged (Figure 3C). However, in type C pattern the nuclear membrane was not observed (Figure 3D), suggesting that a precocious nuclear envelope loss could account for this dispersion of condensed chromatin.
These results led us to determine the state of nuclear lamina in nuclei having different patterns of chromatin condensation. Nuclei from fibers of the outer cortex, without signs of chromatin condensation, exhibited an intact nuclear lamina (Figure 4A). Nuclei having type B condensed chromatin presented striking results. In some cases the nuclear lamina appeared undamaged (Figure 4B, lower nucleus), but in others some discontinuities could be detected (Figure 4B, upper nucleus). Type C pattern displayed different degrees of nuclear lamina degradation. In some cases the lamina exhibited small ruptures and holes (Figure 4C) leaving regions of condensed chromatin exposed (Figure 4Q). In others, besides some small breaks, the nuclear lamina had an irregular profile (Figure 4D) that seemed to delineate the chromatin clumps (Figure 4R). Finally, in fibers only containing nuclear remnants, the nuclear lamina appeared extremely fragmented (Figure 4E,F,G). Immunoreactive lamins appeared or not associated with condensed chromatin (Figure 4S,T,U).
Activity and immunodetection of DNase I in fiber cells
To determine the presence of DNase I in lens fibers we used nuclease activity gel assays, performed in the presence of micro molar concentrations of calcium and magnesium at neutral pH. The analysis of SDS-soluble fractions of cortical and nuclear fibers showed the presence of several Ca++/Mg++ dependent nuclease activities, ranging from 30 to 70 KDa in size (Figure 5A). The smallest activity band revealed by these assays (Figure 5A, arrowhead) exhibited a slightly higher molecular weight than the bovine pancreatic DNase I used as control. To determine if this activity band was DNase I, western blots were made using the polyclonal antibody Ab2983. This antibody, prepared by us, has been previously characterized and it recognizes only one band in bovine pancreas and parotid . In the fiber samples analyzed here the antibody also recognized one band that, though it was slightly heavier than pancreatic DNase I, co-migrated with the parotid enzyme (Figure 5B).
Sub cellular distribution of DNase I in fiber cells
As previously said, we have already shown that DNase I is localized in the cytoplasm of lens epithelial cells, and that the enzyme is confined to secretory pathway organelles . Consequently, once we determined the presence of DNase I in fiber cells, we assessed its sub cellular localization. In situ immunodetections were performed on whole mounts of lens tissue as well as in cryosections. DNase I was localized in the cytoplasm of the most external cells of the proliferation zone (Figure 6A,B,C). It was also cytoplasmic in cells from the meridional rows (onset of fibergenesis, Figure 6D,E,F). In more elongated fiber cells there was immunoreactive enzyme in the cytoplasm, mainly concentrated in close proximity to the cell membrane (Figure 6G, arrow), although DNase I immunoreactivity was also found at the nuclear territory (Figure 6G,H,I). We also found nuclear labeling to be specific, as it disappeared when an a-DNase I depleted antibody was used as negative control (data not shown).
Figure 7 shows fiber nuclei representing the major types of nuclear shapes and chromatin states described above. It can be observed that DNase I exhibits a strong immunoreactivity over the whole nucleus and appears concentrated in highly labeled patches distributed at the nuclear surface. Figure 7A,F,K show a nucleus in a young superficial fiber, whereas the nucleus in Figure 7B,G,L is one in which some chromatin condensation is evident. In both cases there is an immunoreactive ring surrounding the nucleus, an image reminiscent of a perinuclear space labeling. The comparison of DNase I patches and regions of condensed chromatin showed that they did not colocalize (Figure 7A,F,B,G). The association of DNase I and condensed chromatin is very close in collapsing nuclei (Figure 7C,H,M), in fibers where the nuclear material has the distribution named type C (Figure 7D,I,N) as well as in nuclear remnants (Figure 7E,J,O).
Co-localization of DNase I and fragmented DNA
The results presented above demonstrated that DNase I accumulates at the nuclear territory in maturing fibers and suggested that the enzyme was in close proximity to chromatin. To assess whether DNase I could be one of the nucleases generating free 3'-OH ends in DNA (detected by nick-end labeling in degenerating nuclei), cryosections were double labeled by TUNEL assay and by anti-DNase I. Oval nuclei, in which DNase I appeared distributed on the whole nuclear territory and was particularly concentrated in patches, as in Figure 7B, were the first showing a faint TUNEL positive reaction (Figure 8A,F) but the labeling did not co-localize (Figure 8K). However, there was an accurate co-localization in those fibers having nuclear material distributed according to the type B and C patterns already described (Figure 8L,M,N). Nuclear remnants were also highly labeled by a-DNase I antibodies and TUNEL (Figure 8E,J,O).
The first noticeable signs of nuclear breakdown during bovine lens secondary fiber differentiation appear in oval shaped nuclei in which chromatin starts to condense. We described here two new patterns of chromatin condensation (named types B and C) in addition to the classic one described previously [14,15]. Either pattern can be further characterized including the state of the nuclear envelope. In all cases the final stage of nuclear degradation is constituted by small, fully condensed nuclear remnants as found in all species so far studied [14,16-18].
It has been generally considered that chromatin degradation is a late event during nuclear breakdown, occurring once the nuclear envelope is completely or partially degraded. In bovine lens secondary fibers, a TUNEL positive signal has been found in nuclei at the beginning of the round-up process when a large fraction of chromatin is already condensed, as well as in nuclear remnants . In other mammalian species, such as monkey and rat, only nuclear remnants appeared labeled [18,19]. Interestingly, an intense TUNEL label was also found in nuclei exhibiting marginal chromatin in chick embryo lenses . We found that nuclei with fully condensed chromatin (patterns A, B, and C) and nuclear remnants were TUNEL positive. However, some elongated nuclei with only partially condensed chromatin were also labeled by this assay. Our results suggest that in bovine lens secondary fibers the accumulation of this type of DNA nicks starts early, shortly after the beginning of chromatin condensation and before the appearance of noticeable nuclear lamina discontinuities. This last observation differs with previous results obtained in bovine lens in which all TUNEL positive nuclei have the lamina fragmented .
The presence of DNase I in bovine lens fibers was determined by nuclease activity gel and western blot assays. The enzyme isoform present in lens fibers exhibited the same relative molecular size of the parotid one, as we already found in epithelial lens cells , suggesting the possibility that both glycosylation sites (Asn18 and Asn206 in the mature protein) were occupied [21,22]. The presence and subcellular distribution of this enzyme has been previously observed in chick embryo lens where it was found restricted to the nuclear territory of epithelial and fiber cells . Nevertheless, according to our results, in bovine lens fibers the distribution pattern of DNase I changes with the progression of fiber differentiation. DNase I is confined to the cytoplasm in cells that are at the initial stages of fibergenesis, but it is present at cell cytoplasm and nuclear territories in older fibers. The cytoplasmic distribution of DNase I might be explained considering its localization in cells from lens epithelium  and the process of degradation undergone by these organelles during fiber cell differentiation . Its accumulation at the cortical cytoplasm could be the result of its binding to actin filaments once released from the organelles.
As far as we know, this report is the first showing how the nuclear distribution of DNase I evolves from the nuclear periphery to a tight association with highly condensed chromatin during lens fiber differentiation. Furthermore, the co-distribution of DNase I with highly fragmented DNA having 3'-OH-free ends was also shown. However, this co-distribution of DNase I and fragmented chromatin was only observed when almost all chromatin was condensed and TUNEL positive. These results suggest that DNase I might have a role in DNA degradation during the last stages of the process. Therefore, other nucleases generating DNA 3'-OH-free termini should be taken into account. Several calcium/magnesium dependent nuclease activities have been described in lens fibers (review in ). We also found other cation dependent nucleases in bovine lens fibers identified by the dumbbell form of the activity bands and the presence of a dark zone encircling them in zymograms, where we have previously demonstrated the presence of DNA single strand breaks . Two other nucleases have been found in chicken lens and proposed to be involved in nuclear DNA degradation: DFF  and L-DNase II . Only the caspase dependent DFF, present in lens epithelium and fibers, generates the 3'-OH-free ends in the DNA detectable by the TUNEL assay. However, since it is possible that as a consequence of a phosphatase activity the DNA 3'-PO4-free ends be removed and 3'-OH-free ends become exposed, L-DNase II should not be ruled out as candidate. DLAD, which shares 34% identity with DNase II, generates 3'-PO4-ends in the DNA . This enzyme has been recently reported as the nuclease responsible of DNA degradation in mouse lens , a species in which terminal differentiating lens fiber nuclei were not labeled by TUNEL .
Taken together, our results suggested the following sequence of events during bovine lens secondary fiber nuclear breakdown; (a) onset of chromatin condensation, (b) production of DNA breaks having 3'-OH-free ends in condensed chromatin, (c) spreading of condensation and fragmentation throughout the whole chromatin, (d) beginning of nuclear envelope (lamin) degradation and association of DNase I with fragmented chromatin, and (e) appearance of nuclear remnants which remain associated with DNase I. Interestingly, DNase I was found closely associated to fragmented DNA before the detection of nuclear membrane degradation, therefore, further experiments are needed to elucidate the mechanisms for its nuclear translocation.
The authors are very grateful to Dr. Ricardo Benavente, Department of Cell and Developmental Biology, Biocenter, University of Wurzburg, Germany, for the monoclonal antibody L3f4 and Dr. Ignacio Pereira from Frigorifíco Matadero Carrasco (Montevideo, Uruguay), for kindly supplying the bovine eyes. This work was supported in part by PEDECIBA (Ministerio de Educación y Cultura, Universidad de la República), Uruguay.
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