Premeiotic DNA Synthesis and the Time of Chromosome Pairing in Locusta Migratoria Peter B

Proceeding8 of the National Academy of Science8 Vol. 66, No. 1, pp. 94-98, May 1970

Premeiotic DNA Synthesis and the Time of Chromosome Pairing in Locusta migratoria Peter B. Moens

DEPARTMENT OF BIOLOGY, YORK UNIVERSITY, TORONTO, ONTARIO, CANADA Communicated by M. M. Rhoades, February 2, 1970 Abstract. Labeled spermatocytes from locusts killed 0, 1, 2, 3, 4, and 6 days after exposure to tritiated thymidine were examined with the electron microscope to determine their meiotic stage. It was found that tritiated thymidine uptake in young spermatocyte nuclei is completed some 24 hours before synaptinemal complexes are formed. It is concluded that in the locust, premeiotic DNA synthesis is completed well in advance of pairing of homologous chromosomes as marked by the formation of synaptinemal complexes. The G-1 and the S-phase are in the order of 24 hours each, while meiotic prophase lasts four to five days under the conditions used.

The accumulated evidence in plants and animals makes a clear chronological separation between premeiotic DNA synthesis and meiotic chromosome pairing. l-l() In each of these cases, however, the time of chromosome pairing could be determined only approximately because of limitations inherent in the material and in the methods. In fact, the actual pairing of chromosomes is observed only rarely in light microscopy. As a consequence, the general appearance of the nucleus is used as an indicator of the zygotene, or pairing stage of meiotic prophase. For instance, the polarization of chromosome ends and the formation of the "bouquet stage" have been used as such an indicator of the zygotene stage.3' I The discovery of a pairing structure between the synapsed chromosomes at meiotic prophase 1-I3 allows the onset and progression of pairing to be diagnosed accurately at the fine-structure level. The pairing of electron-dense axial cores of the unpaired chromosomes into the synaptinemal complexes of paired chromosomes has been described in detail for Locusta migratoria.14 In this study, labeled spermatocytes from locusts killed 0, 1, 2, 3, 4, and 6 days after exposure to tritiated thymidine were examined with the electron microscope to determine their meiotic stage. Under the conditions employed, it was found that chromosome pairing starts about 24 hours after the completion of premeiotic DNA synthesis. Materials and Methods. A locust population is maintained continuously at York University. There is a 12-hr light period at 370 and a 12-hr dark period at 240C. Male locusts in late fifth or early sixth instar have testes with predominantly spermato- gonia and spermatocytes, and they are therefore suitable for this type of investigation. Such males were given three injections of 0.0025 ml tritiated thymidine (1 mCi/ml spec. act. 5000 mCi/mM), 30 min apart.15 Six locusts were killed half an hour after the last 94 Downloaded by guest on September 26, 2021 VOL. 66, 1970 GENETICS: P. B. MOENS 95

injection. Two locusts were killed on each subsequent day, up to day 6, with the excep- tion of day 5. For light microscopy, part of the testis of each animal was fixed in Kahle's fixative, stained by the Feulgen reaction, and then squashed or embedded in wax. For electron microscopy the remainder of the testis was fixed in 2% glutaraldehyde solution in phosphate buffer for 2 hr, and postfixed in a buffered 2% osmium tetroxide solution for 1 hr. After dehydration through an alcohol series and propylene oxide, individual testicular tubules were embedded in Epon. Longitudinal sections of the tubules, about 800 i thickness, were cut on an MT II Porter Blum microtome with a duPont diamond knife. Several consecutive thin sections were collected on a single hole Formvar-covered grid and the next thick section, about 1/4 to 1/2 u, was deposited on a gelatin-subbed glass slide. The thin sections were stained with uranyl acetate and lead hydroxide. The thick sections on glass were covered with Kodak A.R. 10 autoradiographic stripping film. After 60 days, the film was developed in D 19 for 4 min. Phase contrast photomicrographs of such a thick section showed the position of labeled spermatocytes and these cells were then marked on low magnification electron micrographs of the corresponding thin section. Subsequently the fine structure details of all the cells in the thin section were determined. Results. Each testicular tubule contains most stages of meiosis: proximally are sperm cells and distally are the spermatogonial cells. Groups of an estimated 64 spermatocytes form a cyst and each cyst is surrounded by interstitial cells and by a membrane. Individual cysts can be identified in sectioned light microscope and electron microscope preparations. The cells of a cyst develop nearly synchronously,3' 9, 15 and the nuclei of a cyst are therefore all labeled or they are all unlabeled. In Figure 1, each solid line represents one labeled cyst. The place of the line indicates the general meiotic stage of the cells in the cyst, and the length of the line depicts the range of developmental stages found within the cyst. The developmental stages of cells in unlabeled cysts adjoining the labeled cysts are shown, in summary, by the hatched areas. The number of cells seen in a section through a cyst varies from 3 to 16 with an average of 8. The developing spermatocytes were classified according to the conventional stages of meiotic prophase which are listed across the top of Figure 1. Through the use of the electron microscope, several developmental phases could be dis- tinguished within each stage and these substages are marked by roman numerals. The space assigned to each stage or substage in Figure 1 does not reflect the length of time the spermatocyte spent in that stage, but it does reflect the range of intermediary conditions that could be recognized with the electron microscope. The characteristics of the substages are the following: (I) This stage, the G-1 stage, follows the last spermatogonial divisions and there is no uptake of tritiated thymidine. At first the nuclei are strongly lobate and the cells appear multinucleate in section. The X chromosome has two nuclear membranes around it and appears separate from the rest of the nucleus in sections. The X chromosome becomes more densely stained during G-1, until it is denser than the other chromosomes. The G-1 phase lasts nearly as long as the next S-phase (II + III + IV + V). (II) The nucleus is round, the dense X chromosome is enclosed in two membranes and appears separate from the rest of the nucleus. Thymidine-3H is incorporated at this stage. (III) The X chromosome, surrounded by a membrane, lies within the nucleus. The X chromosome is located at the periphery of the nucleus, usually in a bulge of the nuclear membrane. Thymidine-3H is incorporated at this stage. (IV) The Downloaded by guest on September 26, 2021 96 GENETICS: P. B. MOENS PROC. N. A. S.

STAG[ PREMEITIC INTERPHASE PRE I[PTUIENE LEPTTINE-ZYGOTtNE PACHYTENE MEITPH;i;i iT,. PY I E m i E E M I E m

FIG. 1.-The progression of locust spermatocytes, labeled during premeiotic interphase, through the stages of meiotic prophase. Across the top are the conventional meiotic prophase stages as well as a number of substages which can be differentiated with electron microscopy. The substages, marked by roman numerals, are described in the text. The number of days from the injection with tritiated thymidine to sacrifice are marked in arabic numbers down the side. Each solid bar represents one testicular cyst with labeled spermatocytes. The position of the bar marks the general meiotic stage of the cells in the cyst and the length indicates the range of developmental stages within the cyst. The hatched areas summarize the meiotic stages of unlabeled spermatocytes. The arrows suggest the progression of spermatocyte development through meiotic prophase in time. The observations reported are based on the following material: Day 0, 4 locusts, 2 testicular tubules of each locust, altogether 21 labeled cysts, a total of 167 labeled cells, as well as unlabeled cysts neighboring the labeled cysts. Day 1, 2 locusts, 2 tubules each, 21 labeled cysts, 160 cells, and unlabeled cysts. Day 2, 2 locusts, 2 tubules from one, 1 from the other, altogether 23 labeled cysts, 200 labeled cells, and unlabeled cysts. Day 3, 2 locusts, 1 tubule from each, 13 labeled cysts, 124 labeled cells. Day 4, 2 locusts, 1 tubule each, 26 labeled cysts, 143 cells. Day 6, 2 locusts, 1 tubule each, 14 labeled cysts (excluding spermatids), 124 cells. membrane surrounding the X chromosome is no longer continuous. Thymidine- 3H is incorporated at this stage. (V) Small fragments of the X chromosome membrane are present in all or most of the nuclei of a cyst. Thymidine-3H incorporation is terminated during this stage. (VI) The nucleus has a dark staining X chromosome at the periphery and the rest of the chromatin is evenly distributed throughout the nucleus. Fragments of the X chromosome membrane are still present in some nuclei but they are rare and they are only oc- casionally seen in a single section of a cyst. (VII) Electron dense centers are present in some of the chromosomes. These are the precursors of the cores in the next stage but they lack the continuity of cores. The X chromosome and membrane fragments are as in VI. (VIII) At the leptotene stage, there are electron dense axial cores in the unpaired chromosomes. They are particularly pronounced at the polar region of the nucleus, the region opposite the centrioles Downloaded by guest on September 26, 2021 VOL. 66, 1970 GENETICS: P. B. MOENS 97

and the mitochondrial mass of the cell. (IX) At the zygotene stage the nucleus contains axial cores and sets of paired cores, the synaptinemal complexes. (X) At early pachytene, the formation of synaptinemal complexes is completed and there are no axial cores present in the nucleus. The synaptinemal complexes have broad lateral elements, particularly at the polar region. The chromatin is fairly diffuse. (XI) The chromatin is condensed around the synaptinemal complexes. The lateral elements are not as pronounced as in X. (XII) The lateral elements are indistinct but the central elements of complexes are still present. One set of centrioles remains at the polar position while the other set is found in intermedciate positions on its way to the opposite side of the nucleus. This late pachytene stage probably fuses into diplotene but that dis- tinction is not clear in electron micrographs. The timing of some meiotic events can be deduced from data in Figure 1: (1) The DNA synthesis phase, S-phase, of premeiotic interphase lasts about 24 hours. The youngest spermatocytes which incorporate thymidine-3H are in stage II (Fig. 1). Twenty four hours after treatment (day 1) such cells have progressed to stage V, the end of S-phase. Also, 24 hours after treatment the most advanced unlabeled interphase cells are at stage IV. These cells presumably were at the end of G-1, in stage I, at the time of treatment and therefore did not incorporate thymidine-3H. It follows that spermatocytes require some 24 hours to progress from the beginning of the S-phase to the later part of the S- phase (Fig. 1, hatched area of day 0, stage I, to hatched area of day 1, stage IV). (2) It takes somewhat over 24 hours for spermatocytes which complete premeiotic DNA synthesis in stage V, to reach the stage of chromosome pairing as defined by the formation of synaptinemal complexes at stage IX. Some of the most advanced labeled spermatocytes at day 1, presumably the ones labeled in late S-phase at day 0, have just started to form axial cores in stage VIII (Fig. 1). Pairing of these cores into synaptinemal complexes follows soon hereafter in the locust. Furthermore, the spermatocytes which had just completed the S-phase in late interphase stage V at the time of treatment, and therefore did not incorporate thymidine-3H, have reached the beginning of leptotene after 24 hours (Fig. 1, the beginning of hatched areas at days 0 and 1). (3) Six days after treatment, the labeled spermatocytes were mainly in the meiotic metaphases and telophases, and there were labeled early spermatids. The length of meiotic prophase, from pre-leptotene to metaphase I, is therefore in the order of four to five days. The time from premeiotic DNA synthesis to metaphase I has been reported for several other Orthoptera. For the desert locust Schistocerca gregaria the time is 13 to 14 days at 300C and 6 days at 400C,17 for grasshopper Goniaea australasiae the time is 25 days at 260C and 14 days at 370C,18 and for the grasshopper Romalea microptera the time is 24 days at 20 to 250C.9 (4) The spermatogonial cell cycle is in the order of 24 hours. Ul- timate gonial cells labeled in S-phase at day 0 had entered premeiotic interphase, G-phase, at stage I after 24 hours (Fig. 1, day 1). On day 2, these cells reached stages II and III. On days 3 and 4, cells labeled in earlier gonial cycles also reached stages II and III. A comparable spermatogonial cycle of 1.5 days has been reported in Schistocerca gregaria at 400C.17 (5) Development of sper- matogonia and spermatocytes is not fully synchronous in two aspects: (a) Downloaded by guest on September 26, 2021 98 GENETICS: P. B. MOENS PROC. N. A. S.

Although usually the nuclei of one cyst are all labeled or all unlabeled, occasional cysts have labeled and unlabeled cells. Such asynchrony was also noted by direct observations on differences in developmental stages of the cells of one cyst. For example, in a case where there was one labeled nucleus in a leptotene cyst it was found that the labeled nucleus was structurally in a retarded stage relative to the other cells. Cysts having several labeled nuclei were classified as labeled in this report. (b) The time of the spermatogonial cycle and meiotic development is not rigidly fixed. Animals killed four and six days after treatment had labeled cysts in most stages of meiotic prophase (Fig. 1). The first wave of labeled cysts is clearly defined but the subsequent waves cannot be resolved with the same certainty. Generalizations about meiotic prophase which are well supported by observa- tional and experimental evidence include: (a) Premeiotic DNA synthesis is completed prior to the synapsis of homologous chromosomes.1-10 (b) Crossing over and chiasma formation takes place between synapsed homologous chromosomes.17 20 (c) Synaptinemal complexes are a necessary adjunct of chromosome pairing, crossing over, and chiasma formation.13 16, 20 The direct evidence presented here that synaptinemal complex formation is initiated some time after the completion of premeiotic DNA synthesis in the locust is in agree- ment with these propositions. Essentially the same conclusion is implicit in the reports on DNA synthesis and synaptinemal complex formation in Lilium.20-21 Similar observations in the yeast Saccharomyces cerevisiae22 require clarification of the structures reported to be synaptinemal complexes. Financial support from the National Research Council of Canada, and technical assistance from Mrs. L. Oostwoud are gratefully acknowledged. Dr. B. Loughton of the Department of Biology, York University, supplied the locusts and helped by suggesting the autoradiographic techniques used here. 1 Callan, H. G., and J. H. Taylor, J. Cell Sci., 3, 615 (1968). 2 Das, N. K., E. D. Siegel, and M. Alfert, J. Cell Biol., 25, 38.7 (1965). I Lima-de-Faria, A., J. Cell Biol., 6, 457 (1959). 4Monesi, V., J. Cell Biol., 14, 1 (1962). 5 Plaut, W. S., Hereditas, 39, 438 (1953). 6 Swift, H. H., and R. Kleinfeld, Phys. Zool., 26, 301 (1953). 7 Taylor, J. H., Am. J. Bot., 37, 137 (1950). 8 Ibid., 46, 477 (1959). 9 Taylor, J. H., J. Cell Biol., 25, 57 (1965). 10 Rossen, J. M., and M. Westergaard, Compt. Rend. Trav. Lab. Carlsberg, 35, 233 (1966). 1 Fawcett, D. W., J. Cell Biol., 2, 403 (1956). 12 Moses, M. J., J. Cell Biol., 2, 215 (1956). 13 Ibid., 4, 633 (1958). 14 Moens, P. B., Chromosoma, 28, 1 (1969). 15 Ibid., 19, 277 (1966). 16 Moses, M. J., Ann. Rev. Gen., 2, 363 (1968). 17 Henderson, S. A., Nature, 211, 1043 (1966). 18 Peacock, W. J., in Replication and Recombination of Genetic Material, ed. W. J. Peacock and R. D. Brock (Canberra: Australian Academy of Science, 1968), p. 242. 19 Rhoades, M. M., in Replication and Recombination of Genetic Material, ed. W. J. Peacock and R. D. Brock (Canberra: Australian Academy of Science, 1968), p. 229. 20 Roth, T. F., and M. Ito, J. Cell Biol., 35, 247 (1967). 21 Hotta, Y., M. Ito, and H. Stern, these PROCEEDINGS, 56, 1184 (1966). 22 Engels, F. M., and A. F. Croes, Chromosoma, 25, 104 (1968). Downloaded by guest on September 26, 2021