Electron Microscope Autoradiography of 3H-Thymidine Incorporation during the Zygotene Stage in Microsporocytes of Lily

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1 CELL STRUCTURE AND FUNCTION 3, (1978) C by Japan Society for Cell Biology Electron Microscope Autoradiography of 3H-Thymidine Incorporation during the Zygotene Stage in Microsporocytes of Lily Nori Kurata and Michio Ito* Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University, Fukuoka 812 and *Department of Biology, Faculty of Science, Nagoya University, Nagoya 464 ABSTRACT. The site of chromosomal DNA synthesis during zygotene stage of meiosis was explored by electron microscope autoradiography. Zygonema of Lilium longiflorum cultured in the presence of 3H-thymidine for 24 h frequently showed label distribution over the axial cores prior to formation of synaptinemal complexes. When the culture was continued in isotope-free medium, the zygotene label appeared over synaptinemal complexes. Light microscope autoradiographs of cells showed the zygotene label over both paired and unpaired regions of chromosomes at pachytene. The relevance of these observations to the formation of synaptinemal complex was discussed. Synthesis of a small fraction of nuclear DNA has been demonstrated during the zygotene stage in microsporocytes of liliaceous plants (3, 4). Although zygotene DNA in the lily constitutes only about 0.3 % of the total genome, the timing of its synthesis has been shown to be significant to the behavior of chromosomes during the zygotene stage (6, 11, 12). Application of an inhibitor of DNA synthesis to leptotene cells interfered with the entrance of these cells into the zygotene stage and with the pairing of homologous chromosomes (6). Inhibition during the zygotene stage interrupts formation of the synaptinemal complex (SC) without destroying the complex already formed (11). The site of synthesis of zygotene DNA has been identified by autoradiography and shows that there is a general distribution of the lable over various parts of metaphase chromosomes in cells exposed to 3H-thymidine (3H-TdR) during the zygotene stage (5). Incorporation of a small amount of label into the nuclei during meiotic prophase has been also demonstrated in several publications (8, 9, 14). This shows how widespread this phenomenon is in meiotic division. On the basis of these observations, a close relationship appears to exist between DNA synthesis during the zygotene stage and the process of synapsis, and the speculation that zygotene DNA molecules are present in the SCs of the chromosomes is circumstantial evidence for this relationship. We therefore directly tested the speculation with cultured microscporocytes of Lilium under electron microscope autoradiography. The experiments were performed to determine the site of incorporated 3H- TdR into chromosomes during the synaptic process. 349

2 350 N. Kurata and M. Ito MATERIALS AND METHODS Meiotic cell culture. Lilium longiflorum var Hinomoto was used as the source of microsporocytes for autoradiography and var Georgia was used for the cytological studies. No cytological differences were observed between the two varieties. Microsporocytes were obtained from buds 12 mm (early leptonema) and 13 mm (middle leptonema) long. Extruded cells were cultured essentially as described previously (7) except that all cultures were maintained at 25 Ž. Although meiotic cells were extruded from a bud as a total of 24 identical strings, the strings of meiocytes from an individual bud were cultured separately. For autoradiography, cells were first cultured for 24 h in the basic medium supplemented with 2 mm deoxyadenosine (AdR) to arrest DNA synthesis during the zygotene stage (6). Following this culture period, meiocyte strings were transferred to supplemented medium containing 20 ƒêci/m1 of 3H-TdR (spec. act. 6 Ci/mM) and 0.5 mm of unlabeled uridine, the latter helping to reduce the incorporation of thymidine catabolites into RNA (5). Incubation was continued for 24 h. For zygotene cells, 24-h intervals were found to be the optimum period for intense labeling within the cells (5). After the incubation period, meiotic cells were returned to the basic medium without 3H-TdR and cultured again for 1 to 2 days. Part of cells from each bud was cultured in the basic medium without AdR as the control. Part of the post-culture after 3H-TdR incubation was prolonged until meiosis was complete. Cytological observations. At the onset of the experiment and on each of 5 successive days, one string from each bud was used for a chromosome squash preparation to determine the stage of meiosis and the degree of survival of the cells. Light microscope autoradiography. At the end of incubation with 3H-TdR and on the 1st day of the post-culture, two strings from each bud were fixed in 1:3 acetic alcohol for light microscope autoradiography, and after Feulgen staining each string of meiocytes was squashed on a slide. The cells were coated with Sakura NR-M2 emulsion and stored for 1 month at 4 Ž. After developing a coat with D-19 developer at 20 Ž for 4 min, the slides were made permanent with Euparal. Standard procedures were used for the DNase and RNase treatments (2). Electron microscope autoradiography. At the onset of incubation with 3H-TdR and on each of 3 successive days, electron microscopy was performed with meiotic cells fixed first in 3 % glutalaldehyde then post-fixed in 1 % osmium tetraoxide. The cells were then embedded in Epoxy resin and thin sections were stained with a 2 % solution of uranyl acetate. For autoradiography, thin sections were coated with Sakura NR-H2 emulsion by the wire loop method, and stored for 1 month at 4 Ž. Development with Konidol-X at 20 Ž for 4.5 min and fixing with Konifix were carried out as usual. To remove jellatin and to stain with lead monoxide, sections were treated for 1 h in a 20-fold dilution of a medium containing 4 g of sodium hydroxide and 1.6 g of potassium-sodium tartrate in a saturated solution of lead monoxide. All sections were washed thoroughly in CO2-free distilled water and viewed with JEM-7A electron microscope. The distribution of silver grains in the electron microscope autoradiographs was calculated for at least 20 thin sections in each cell component. The values obtained in this study are ratios of the grains per unit area, represented as concentrated because the number of grains counted in each section was too small. RESULTS Cytology in AdR treated cells. Strings of meiotic cells placed in culture at the leptotene stage in the presence of AdR had no structures that resembled the axial core or

3 Autoradiography in Meiotic Lily Cells 351 the SC for 24 h. In the absence of AdR, cells cultured from the same bud proceeded from the leptonema to the zygotene stage in the same time interval had these structures. These results show that cells treated with AdR at the leptotene stage, prior to the onset of zygotene DNA synthesis, do not from the axial core and that further progression from the late leptotene stage is arrested for the intervals of the exposure to the inhibitor. When the inhibitor was removed after a 24-h treatment, the behavior of chromosomes in the meiocytes during subsequent culture seemed to be normal. After 1 day of culture, chromosomes in the squash preparation had initiated pairing, and nuclei which frequently exhibited axial core-like structures with occasional SCs were observed under the electron microscope. In a cross section of single chromosomes identification of the axial core is ambiguous because little difference can be seen from the surrounding chromatin in density and in structure, but the structures may be presumed to exist because of their longitudinal continuity through the chromosomes. When culture was prolonged, the chiasma formation at the diplotene stage and the metaphase configuration as well as the rate of meiosis appeared to be the same as in cells under in situ conditions. AdR treatment was also effective for maintaining synchrony in the cultures, in terms of the frequency of metaphase I cells, and it caused a decrease in the ratio of abnormal tetrad configurations. Thus a 24-h treatment with AdR keeps all cells at the late leptotene stage in which adaptation of the cells to a synthetic medium in vitro is completed. After the removal of the inhibitor the cells appear synchronously to start zygotene DNA synthesis accompanied by pairing between homologous chromosomes. Light microscope autoradiography. To test the labeling of chromosomes by exogenously supplied 3H-TdR in zygotene meiotic cells, as demonstrated by Ito and Hotta (5), in more detail autoradiographic analysis was carried out using squashed preparations under a light microscope. Two kinds of cells, one fixed immediately after 24 h of culture in 3H-TdR and the other fixed after the post-culture in unlabeled medium for 24 h, were examined for pairing status and the pattern of the label over the nuclei. The former had thick threaded regions where the homologs appeared to be paired in about half of chromosomes. Silver grains were more or less homogeneously distributed over all of the chromosomes, in both the thin and thick threads, but there was no cluster of grains within the nuclei (Fig. I a). The cells post-cultured for 24 h had thick threads of chromosomes which seemed to have fully completed synapsis and thus were in the pachytene stage. The majority of chromosomal label was DNase sensitive and a very small amount of the radioactivity was removed with RNase. The number of grains counted on 20 nuclei was decreased by about 10 % with RNase treatment, and there was no localization of the reduction in a specific region of chromosomes. The behavior of cells on enzyme treatment are consistent with results reported previously (5). The DNase sensitive grains were retained over all the chromosomes with unpaired regions which were occasionally observed (Fig. 1 b). This labeling pattern suggests that zygotene DNA synthesis occurs before the pairing of homologs. Electron microscope autoradiography. Zygotene nuclei fixed immediately after incubation in the presence of 3H-TdR for 24 h frequently showed longitudinal structures that resemble the axial core. A few chromosomes completed synapsis and had SCs embedded in some masses of chromatin. The most important observation on the labeling of zygotene chromosomes after 24 h of incubation in the labeled precursor is shown in Fig. 1 c and 2. As described earlier, most of the nuclear label is removed with

4 352 N. Kurata and M. Ito Fig. 1. Autoradiographs of nuclei at the zygotene and early pachytene stages cultured for 24 h during the zygotene interval in the presence of 3H-thymidine. a, zygotene nucleus fixed immediately after the culture period; b, part of a nucleus post-cultured for 24 h in the isotope-free medium to the early pachytene stage; c, electron micrograph of a cell fixed immediately after labeling. It is difficult to identify the axial cores in the cross section of the chromosomes, but may be assumed to exist because of the longitudinal continuity through the chromosomes. Silver grains are present over the presumed axial cores.

5 Autoradiography in Meiotic Lily Cells 353 Fig. 2. Same as in Fig. 1. a, fixed immediately after labeling. b, post-cultured for 24 h. Synaptinemal complexes are seen surrounded by masses of chromatin. C, same as in b, but the lateral elements of the synaptinemal complex are partially identified.

6 354 N. Kurata and M. Ito TABLE 1. GRAIN DISTRIBUTION IN AUTORADIOGRAPHS OF ZYGOTENE CELLS LABELED WITH 3H-TdR Meiotic cells were obtained at early or middle leptotene, cultured for 24 h with AdR as described in METHODS, then labeled for 24 h in the presence of 3H-TdR. The cells then were fixed immediately for autoradiography in the electron microscope. Data were calculated from at least 20 thin sections of each particular cell component. Under these labeling conditions the cell wall usually had appreciable amounts of radioactivity (5). Since it is difficulto define precisely the axial core, especially a cross-sectioned one, the representation of grain distribution for condensed chromatin does not discriminate between structures that resemble the axial core and the dense structure of the chromatin. DNase. The number of DNase-sensitive grains counted in each of the sections is too small to allow conclusions about the possible localizations of the label because of the small amount of DNA synthesis in the zygotene stage and because of the thin sectioned nuclei. However, data on the distribution of grains over each of the cell components provided evidence that the label is concentrated over the dense structure of the chromatin (Fig. 1c). Table 1 shows the frequency of grain distribution in zygotene cells cultured for 24 h in presence of 3H-TdR. Most of the label is present in the presumed axial cores, or in the chromatin next to the structures (Fig. 2a). However, whether the longitudinal structures seen in Fig. 2a are homologs already juxtaposed, or are single axial cores is not certain. In cells post-cultured in unlabeled medium for 24 h, the average number of SCs seen in the sections through the nuclei noticeably increased. Autoradiographs on these nuclei showed that most of the nuclear label was in the synaptic regions of the chromosomes (Fig. 2b and c). The frequency of grain distribution over the SCs was as much as ten fold that over the other chromatin area. The finding that the axial core produces a lateral element may account for the grains present over the SCs. Thus, we concluded that chromosomal DNA synthesized during the zygotene stage is located in the lateral elements of the SCs, and that DNA synthesis is required to initiate formation of the SC. As reported previously (5), cytoplasmic labeling in zygotene meiocytes by exogenously supplied 3H-TdR is unavoidable with a prolonged incubation, such as 24 h (Table 1). In addition, the cell wall usually incorporates the methyl label of thymidine into various substances. However, this study does not treat the incorporation of this label. DISCUSSION It is well-known that the synaptinemal complex could be involved, as a unique structure associated with chromatin during synapsis, in bringing the two homologs together in a site-for-site synapsis preparatory to crossing over (10, 13). The demonstration of a small amount of chromosomal DNA synthesis at the time of synapsis (3, 5). combined with evidence that the presence of an inhibitor of DNA synthesis interferes with

7 Autoradiography in Meiotic Lily Cells 355 the formation of the SC (11), strongly indicates that some DNA synthesis is necessary for the formation of the SC. Thus, there is little question that this newly synthesized DNA is intimately involved in the morphogenesis of the SC, but we know virtually nothing of the macromolecules present in the unique structure visible between the paired homologs. Questions remain whether the chromosomal DNA synthesized during the zygotene stage is present in the SC and, if so, when it is replicated during the morphogenetic process of chromosomes. The autoradiographic experiments presented here provide a satisfactory answer to these questions, and it is likely that the DNA is present in the synaptic regions of the chromosomes. Synthesis is localized, to a large extent, in the SCs or in the chromatin next to the SC. Grains appeared first over the axial cores formed in the chromosomes prior to pairing. There was no direct evidence that the grains over the axial cores were derived from the isotope incorporated into the DNA of the cores. However, DNase-sensitive grains were distributed of at a significantly high frequency over the axial cores as compared to the small number of grains on the areas of the surrounding chromatin. Furthermore, the supposition that a 24-h culture period in 3H-TdR medium would obscure the site of DNA replication with products of thymidine catabolism is refuted by the views of Ito and Hotta (5). Thus, we favor the somewhat circumstantial interpretation that most of the radioactivity over the SCs comes from the tritium incorporated in the DNA constituting the axial cores. At least, the zygotene DNA replication is intimately involved in the formation of the SC and we are confident that these studies, together with those previously reported (5), establish the actuality of a small amount of DNA synthesis in the chromosomes of zygotene cells. In spite of our new evidence, we have no answer as to what role DNA plays in the formation of the SC. The incorporation of label into nuclei during meiotic prophase have been observed in several eukaryotes (8, 9, 14), and various speculations have been on made the function of DNA (12, 13). The faint, thin fibers bridging the SC, visualized in the early study by Coleman and Moses (1), may represent DNA. One attractive possibility is that the zygotene DNA is present in special regions of the axial cores that later function as matching sites for the synaptic alignment of homologous segments of chromosomes (12). Since synapsis represents, not total DNA-DNA alignment, but special regions of pairing between homologous chromosomes, the DNA itself may be the species of macromolecule making up the individual synaptic sites for the matching of homologous DNA stretches. Our results demonstrate that zygotene DNA synthesis is required, prior to completion of the SC, during the production of the axial core before production of the lateral elements of the SCs. Light microscope autoradiographs also show grains overlying unpaired regions of the chromosomes. These autoradiographs offer new, direct evidence on the correlation of the timing of DNA synthesis with chromosomal synapsis. In the synaptic process, the juxtaposition of the homologous segments of chromosomes is a transient event which can lead to synapsis and must precede the processes that stabilize pairing in forming SC, which is the final product of the matching process. A simple interpretation of this observation is that zygotene DNA replication is necessary for the juxtaposition of the matching sites, even if the DNA is an essential component of the SC. However, it is not possible to verify this interpretation until more is known about the synaptic event leading to the formation of the SC. An attempt to define more precisely the mechanisms involved in the pairing processes is in progress.

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