ISSN 1990519X, Cell and Tissue Biology, 2012, Vol. 6, No. 3, pp. 280–292. © Pleiades Publishing, Ltd., 2012. Original Russian Text © V.V. Klymenko, Liang Haoyouan, 2012, published in Tsitologiya, 2012, Vol. 54, No. 3, pp. 218–229.
Chromatin in Diapause of the Silkworm Bombyx mori L.: Thermal Parthenogenesis and Normal Development V. V. Klymenko* and Liang Haoyouan Faculty of Biology, Karazin Kharkiv National University, Kharkiv, Ukraine *email:
[email protected] Received September 12, 2011
Abstract⎯By using hematoxylin staining, peculiarities of chromatin of diapausing silkworm embryos were studied in normal and parthenogenetic development. A direct correlation was revealed between the number of interphase chromatin grains and the number of chromosomes in the nucleus; polyploidization of cells in embryo was studied at the stage of diapause. The polyploidization in parthenogenesis is not restricted to endomitotic chromosome set doubling, as it includes 6nnuclei. To explain the more diverse spectrum of polyploid cells in parthenogenesis (as compared with the norm), it is necessary to take into account fusion of cleavage nuclei, which is realized by the cytoplasmic mechanism of karyogamy in the absence of fertilization. On squash preparations, for the first time, we identified primordial germ cells of the diapausing embryo, which are characterized by less compact chromatin, especially in the zygotic variant of development; by larger nuclei and cytoplasm; and by an irregular number and size of nucleoli. Estimation of ploidy of the primary germ cells in parthenogenesis by counting “loose” chromatin grains in diapause is possible and indicates polyploidization in the primordial germ cells. This explains an inevitable admixture of tetraploid eggs in the grain of diploid parthenoclones and its absence in normal development. The cytological method used has revealed a spiral arrangement of chromatin grains on the internal nuclear surface at different ploidy levels. Keywords: interphase, heterochromatin, bisexual development, parthenogenesis, somatic polyploidization, germ line, silkworm, polyploidy, diapause. DOI: 10.1134/S1990519X12030054
INTRODUCTION Diapause in the silkworm takes place in the lifecy cle of mono and bivoltinic breeds. It is convenient for study of a wide spectrum of topics in insect develop mental biology and physiology. Entrance into the dia pause of fertilized and nonfertilized thermoactivated eggs (Astaurov, 1940) 2–3 days after their activation is due to action of the diapause hormone produced by suboesophageal ganglion and entering into maternal organism oocytes (Hasegawa, 1957; Imai et al., 1991). The frequency of cell division falls rapidly, and a long period of rest (up to ten months) occurs in develop ment at the minimal level of metabolism. However, diapause can be prevented if developing eggs are treated at the proper time with hydrochloric acid at a certain concentration; otherwise, the grain entering diapause again acquires the ability to develop at 25°C only after 100–120 days of “wintering” in a refrigera tor (Tazima, 1978). The embryos entering diapause are at the same development stage, the cell cycle in all cells being Abbreviations: CG—chromatin grains, PGC—primordial germ cell.
arrested at interphase. Staining of fixed material with hematoxylin prepared in propionic or acetic acid and squashing down of the embryo in acetochloralhydrate allow obtaining squashed preparations with signifi cantly enhanced staining of the nucleoli, heterochro matin, and nuclear envelope. In such preparations, there was determined in diapause the number of cells in embryos of different ploidy during two types of reproduction: in thermal parthenogenesis by Astaurov (1940) and in normal fertilization. The compared vari ants of development turned out to differ essentially, as, at the same ploidy level, the number of cells in the zygot ically diapausing embryos was almost 1.5 times higher than in the parthenogenetic ones (Klymenko, 1974). Recently, we returned again to interphase hetero chromatin of diapause for a comparative study of poly ploidization in the soma and the germ line of embryos obtained by normal fertilization and thermal parthe nogenesis. The regularity of heterochromatinization in interphase cells is manifested in particular as the obvious similarity of clumps (granules) of heterochro matin and their amount in different nuclei of the dia pausing embryo. In the present work, by using the number of chro matin granules (CGs) in cell nuclei to estimate the
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ploidy level, we evaluated and compared the process of polyploidization in the two abovementioned variants of development and revealed its peculiarities in par thenogenesis. There were also revealed peculiarities of morphology of heterochromatin in diapausing inter phase nuclei of different embryo tissues and of its germ line. The state of heterochromatin at this period reflects not only the physiology of the diapausing embryo, but also the spatial DNA organization in the nucleus directly before the cell entrance to mitosis after the end of diapause. MATERIALS AND METHODS The collection of silkworms of Germ and Stem Cells Laboratory at Karazin Kharkiv National Univer sity was used in this work. Preparations were made by using diapausing grain of diploid clone P29 and tetra ploid clone P4n17 obtained by Astaurov (1968, 1973), as well as grain of new triploid parthenoclones. To study zygotic embryos, we used eggs of the C5 line labeled by sex at the egg stage (Strunnikov and Gula mova, 1960), as well as other laboratory lines. Methods of fixation, staining, and preparing squashes have been described in detail earlier (Kly menko, 1972; Shchegelskaya et al., 1986) and tested successfully on several animal and plant subjects. As a stain, hematoxylin was used (Merck, Germany) pre pared in acetic (Smirnov, 1968) or propionic acid (Henderson and Lu, 1968); as medium for squashed stained material served acetochloralhydrate (Kly menko, 1972). The convenience of this procedure consists in that even an overstained fixed material retained for long enough in acetochloralhydrate can be brought to the desired degree of clearing, the stud ied embryo part isolated from it with microneedles, acquiring in acetochloralhydrate a consistency conve nient for obtaining of monolayer cell preparations. After accurate bordering of coverslips with nail polish, the obtained temporary preparations can be preserved for more than a week and, as a rule, do not deteriorate. The thickness of the preparation cell layer can be var ied by choosing the necessary drop volume, in which an embryo part is squashed. To obtain total prepara tions, a thick layer (the chamber) was obtained by placing plasticine clay on coverslip corners before placing it on the acetochloralhydrate drop with the studied object. RESULTS Determination of ploidy by counting CG in the cell nucleus. In thin squash preparations, cell nuclei of the same ploidy look equal in size; they contain similar heterochromatin granules (clumps); and they often lie separately; therefore, it is not difficult to count them. A count of CG in nuclei of diploid embryos has shown the number of granules per nucleus to be equal to the diploid chromosome number that is 56 in the domestic CELL AND TISSUE BIOLOGY
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silkworm. Deviation from this diploid number as a rule amounts a few granules, which cannot affect the esti mation of ploidy at the haploid chromosome set (n = 28). In the same way, we have made sure that, in trip loid and tetraploid embryos, the amount of CG in nuclei of the overwhelming number of cells corre sponds to the triploid (3n = 84) and tetraploid (4n = 112) sets. A convenient criterion of right estimation of the ploidy level is the number of nucleoli in the nucleus, which corresponds to the number of haploid chromosome sets. Nucleoli can be fused, then a decrease of their number is compensated by an increase in size of visible nucleoli, whereas the total nucleolar volume remains unchanged. Variations of the number of nucleoli in the nucleus are presented in Figs. 1 and 3 at different ploidy levels. Information on the variation of the interphase number CG in diploid, triploid, and tetraploid cells is provided by histograms constructed for samples of 100 cells of embryos of the same ploidy level: the modal classes include more than half of the entire sample, deviations from the modal chromosome num ber are insignificant and cases of deficiency are more frequent than those of excess (Fig. 2). To determine the embryo ploidy, naturally, a dozen cells is sufficient and, after some experience, a look at the preparation under the microscope is enough to establish by the number of CG and nucleoli and the size of nucleoli whether the ploidy level of the studied embryo is different from the diploid one. Such a tech nique of ploidy evaluation is very convenient for experimental obtaining of polyploids in the silk worm—more specifically, in its monovoltine and bivoltine varieties, in which diapause takes place. An amount of CG in the nucleus that is lower than the modal number is easily explained by fusion of granules or by exposure of one under the other, whereas we are inclined to explain a somewhat higher number of CG by the seen granule’s possibly consisting of two or three combined parts, rather than by assuming its sep aration into individual parts due to squashing or by suggesting aneuploidy. Indeed, upon staining with aceto or propionohematoxylin for 1 h at 60°C, chro matin begins to reveal its more complex structure. Based on the similar asymmetry of the presented his tograms (deficiency of granules is present more often than excess), it can be concluded that masking of some granules by others is present markedly more often than is manifestation of their combined structure. The chromatin granules greatly resemble mitotic chromo somes (Astaurov, 1968); however, mitoses and meioses at the studied stage are absent, which corresponds to the presence of nucleoli and nuclear envelopes in nuclei. Somatic polyploidization in early silkworm develop ment. Many insect organs and tissues are known to consist of polyploid cells. Somatic polyploidization begins as early as in embryogenesis and at the larval period in some organs, for instance, in the silk gland,
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(a)
2n (b)
3n (c)
4n
embryo (2n)
(d) Fig. 1. Chromatin granules and nucleoli in embryonic cells of different ploidy (2n, 3n, 4n). (d) part of squash preparation of diploid embryo; obj.: 100× (a–c) and 20× (d).
Number of cells, % 100 90 N = 100, 2n 80 X = 55.5, N4n = 100, 2n 70 Mod = 56 X4n = 111.9, N3n = 100, 2n 60 Mod4n = 112 X3n = 83.1, 50 Mod3n = 84 40 30 20 10 0 … 50 52 54 56 58 60 62…78 80 82 84 86 88 90…106 108 110 112 114 116 118 … Number of chromatin grains per nucleus
Fig. 2. Distribution of embryonic cells on number of chromatin granules (CG) per the nucleus for di (2n), tri (3n), and tetra ploid (4n) clone. N2n, N3n, and N4n—number of cells in the sample from di (2n), tri (3n), and tetraploid (4n) clone, respectively; X2n, X3n, and X4n—the mean number of CG per nucleus in the corresponding sample; Mod2n, Mod3n, and Mod4n—modal classes of CG for the clone 2n, 3n, and 4n, respectively.
it reaches enormous levels (Klymenko and Spiri donova, 1974). The approach that we used in the present work has allowed to estimate this process by the moment when the embryo enters deep diapause. In the review preparations, even at low magnification, among the mass of embryo cells, larger cells are clearly
seen with nuclei of larger diameter; their ploidy was determined by counting CG (Fig. 3). For fast comparison of polyploidization in parthe nogenetic and zygotic variants of development, on squash preparations, we estimated the amount of cell polyploidization in four embryo regions containing in CELL AND TISSUE BIOLOGY
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4n
8n
6n 4n 4n (a)
6n 4n
2n 8n
(b)
2n
(c)
2n
2n
(d)
2n
Fig. 3. Squash preparation of diploid embryo of parthenoclone P29 (×20), on which ploidy of the larger cells (b–d) is determined by counting CG. Obj.: 20× (a) and 100× (b–d).
total about 1000 cells. For this, the embryo was sepa rated with microneedles into four approximately equal parts, in which the cell polyploidization was estimated by counting CG (Table 1). By the same way, the polyploid cells were counted in embryo of the fertilized egg (line re971), i.e., in zygotic development. The results turned out to be dif ferent: polyploid cells were present much more rarely (Table 2). The much rarer presence of polyploid cells in the zygotic variant of development is easily detected. In ten additionally screened diapausing embryos from fertilized eggs, we failed to find cells with ploidy higher than 4n; even tetraploid cells in this development vari ant occur more seldom (viz. Tables 1 and 2, Figs. 3 and 4). From this, the suggestion follows that 6n cells that cannot arise by endomitotic doubling of the chromo some set in the nucleus are formed only in partheno genetic development. Due to the importance of this conclusion, a total of 13 parthenogenetic embryos from P29 clone were studied, each of them being divided for convenience into five approximately equal parts (Table 3, Fig. 5). The number of cells in the embryo at this stage amounts to about 6000; this esti mation is close to what we obtained earlier for other diploid parthenoclones (Klymenko, 1974). In Table 3, CELL AND TISSUE BIOLOGY
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alongside polyploid cells, there are primordial germ cells (PGCs) that will be discussed below; they were found in the amount of 2 to 16 (on average, 5–6) in all embryos except for one (embryo 7), in which we failed to detect polyploid cells. In embryo 5, no polyploid cells were revealed as well, but, in sector d, we found seven primordial germ cells. It is to be noted that PGCs are present most often in sectors c and d (about 90% of their total amount), but are not found in head sector a. Octoploid cells are present more often in sec tors a, c, and especially e (34.8%). Distribution of Table 1. Somatic polyploidization in various areas of dip loid parthenogenetic embryo (clone P29) Cell ploidy 2n 4n 6n 8n
Portion of cells (%) of indicated ploidy in embryo area: a (200)
b (250)
c (400)
d (150)
98 2 0 0
97 3 0 0
94 5 0.75 0.25
96.4 3 0.6 0
Note: In parentheses is the volume of the sample of embryonic cells. The same in Table 2.
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Table 2. Somatic polyploidization in various areas of dip loid zygotic embryo (line re971) Cell ploidy 2n 4n 6n 8n
Portion of cells (%) of indicated ploidy in embryo area: a (257)
b (272)
c (791)
d (311)
98 2 0 0
98 2 0 0
98.5 1.5 0 0
98.5 1.5 0 0
hexaploid cells is shifted toward the head embryo part, and about 60% of their total number are located in sectors a and b. It should be added that, in 19 simulta neously studied diploid zygotic embryos, we failed to reveal both hexaploid and octoploid cells. The tetrap loid cells rather common at this development stage in the zygotic variant are present less commonly than in the parthenogenetic variant; earlier, it was also shown that the number of cells in diapausing embryos was sig nificantly higher in the first than in the second variant (Klymenko, 1974). The more frequent cell polyploidization in the par thenogenetic embryo as compared with the zygotic one seems to be a consequence of the action of some additional mechanism of the formation of polyploid cells, which is peculiar to parthenogenesis. The pres ence of 6n cells in the embryo body makes it possible
to suggest that the assumed mechanism is fusion of the nuclear material of two (2n + 4n) or three (2n + 2n +2n) neighbor cells in the course of embryonic develop ment. As a consequence of this suggestion, in diploid parthenoclones should be present in the grain not only tetraploid eggs, which were discovered as long ago as by Astaurov (1940), but also hexaploid eggs that so far have not been discovered in diploid clones, most likely because they do not differ in size from the diploid ones and are much less frequent than the tetraploid ones. Extraembryonic cells. Extraembryonic cells appear as a result of separation of the embryo from the blasto derm and the remaining cells inside the egg (vitelloph ages). The blastoderm part remaining on the egg sur face gives rise to the monolayer serosa, whereas the rest, which passes into the egg together with the embryo and is closed above it, forms the monolayer amniotic membrane. The cells not belonging to the above membranes and embryo are called vitellophages; by diapause, the vitellophage nuclei turn out to be in the center of yolk spheres filled with yolk globules. Nuclei of amniotic cells are flat and often larger than nuclei of embryo diploid cells (Fig. 6); also present among them are cells the nuclei of which exceed the embryo tetraploid nuclei (Fig. 6: amniotic cells (b, c) as compared with the embryo cells 2n and 4n). In all cases, CG of amniotic nuclei are much smaller than CG in embryonic cells; the correlation of their number with ploidy so far has not been studied.
4n
Fig. 4. Typical preparation of diploid zygotic embryo of the re971 line with two tetraploid cells (4n) in the center. CELL AND TISSUE BIOLOGY
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(b)
(c)
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(d)
(e)
Fig. 5. Embryo of parthenoclone P29 in diapause. Five areas are shown in which polyploid and germ cells are counted for Table 3. Obj.: 10×.
ec ac ec 2n ac ac 4n ec ac
4n (a)
(b)
(c)
Fig. 6. Amniotic cells (ac) and large granular embryonic cells (ec) from different preparations. (a, b) several overstained preparations showing the monolayer amniotic membrane and chromatin fine granularity in nuclei of its cells; (c) normal preparation for comparison of chromatin in amniotic cell (ac) and embryonic cells (ec) 2n and 4n. Obj.: 40× (a) and 100× (b, c).
If not to take into account the large size of serosa cells and to decolor their pigment in the same way as with recessive mutation w2 in the homozygous state (Fig. 7d), they become very similar to amnion cells (by the fine chromatin granularity in their nuclei and by the resulting difficulty in determination of ploidy by the number of CG. Similarity in the CG morphology CELL AND TISSUE BIOLOGY
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can be considered as confirmation of their origin from the same source—the blastoderm not included in the embryo composition. The distribution of pigment in serosa cells and the position of the nucleus in them are of interest for evaluation of the grain physiological state in the course of diapause. It is to be noted that the serosa pigmentation can be darkgray, purple, red, or
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(a)
(b)
(c)
(d)
Fig. 7. Cells of serosa under egg chorion without staining (a) and stained cells (b, c). d—fine granular chromatin of nuclei of serosa of the C5 line deprived, due to mutation, of pigment in the cells of serosa. Obj.: 100× (b, c).
greenishgray or absent altogether, as in the case of the mutation used in the present work (Fig. 7d). The vitellophage nuclei occupying the centers of yolk spheres merit separate study: their chromatin is arranged irregularly, and they can be fragmented, with the character and degree of the fragmentation varying. Nuclei of vitellophages are a separate point worth studying more carefully Fig. 8). Germ cells. One goal of this work was to search for PGCs in the diapausing embryo. Based on histological preparations of Japanese authors (Miya, 1958), at the stages closest to diapause, PGCs differ from somatic cells not only by size, but also by structure of nucleus and cytoplasm. In the region c of the diapausing embryo, we found cells differing from all those pre sented above and identified them as PGCs (Table 7). These cells are characterized by a larger size both of the cytoplasm and of the nucleus; however, in this case, the larger size of the nucleus corresponds to the more active chromatin state rather than to the greater ploidy, this chromatin being significantly more decompactized than that in somatic cells. Therefore,
ploidy estimation by counting CG is very difficult and sometimes virtually impossible. The more active state of PGC chromatin also corresponds to the larger nucleoli, often of unequal size and density, in the amount of one to three or more, but then smaller; it can be suggested that, in these cells, there occur both fusion and fragmentation of the initial number of nucleoli in the nucleus. Although PGCs were revealed both in parthenogenetic (Fig. 9) and in zygotic mate rial (Fig. 11), it was easy to note that, in the case of the latter material, PGC chromatin decompactization in diapause was more pronounced than in parthenoge netic development. We tried to estimate the number of CG resembling partly despiralized chromosomes in germ cells of the studied parthenogenetic embryos (Fig. 9). The distri bution of 60 PGCs on the number of decompacted CG (Fig. 10) confirms the presence of tetraploid cells among them (Fig. 9), which corresponds well to the inevitable admixture of tetraploid eggs in the grain of diploid parthenoclones (Klymenko and Spiridonova, 1977). In the zygotic variant, such analysis turned out CELL AND TISSUE BIOLOGY
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n
n
n n n
n n n
(a)
(b)
Fig. 8. Total preparation of silkworm egg cleaned of chorion, stained with acetohematoxylin, and cleared in acetochloralhydrate. (a) embryo inside the egg in surrounding of yolk spheres (obj.: 10×); (b) yolk spheres at large magnification (obj.: 40×), the center of the vitellophage yolk sphere is occupied by the nucleus (n), the shape of which greatly varies.
to be impossible due to a much more disperse state of the germ cell chromatin in this case, which is obvious from Fig. 11. Distribution of CG in the nucleus. The regular dis tribution of chromosomal DNA in the nuclear volume at different cell cycle stages hardly is more astonishing than the random life origin; however, the unexpected and simple confirmation of the nonrandom distribu tion of chromosomes (chromatin) in the nucleus, which we found in the silkworm diapausing embryo, confirmed once more our conviction that regular dis tribution of genetic material in the nucleus is a neces sary condition of its functioning. Squash preparations can be of different thicknesses. In the thicker layer between the slide and the coverslip, the cell nuclei pre serve their spherical shape, while the CG arrangement in the nuclear volume is closest to its vital state. Spiral ity is detected in the spatial arrangement of the dia pause chromatin. This is easy to ensure by screening the “luckily” oriented (Fig. 12) or “luckily” squashed nuclei. In the latter case, spirality is shown in the dip loid nucleus, in which the helix axis is perpendicular to the preparation plane (the first nucleus); the second and third nuclei confirm the presence of spirality at the triploid and tetraploid levels, respectively (Fig. 13). Not only the helical arrangement of CG on the internal nuclear surface from one pole to the other is worth noting. It can also be noted that, on rather expanded stretches of the helix (its fragments) appear ing upon squashing of the nucleus, granules of identi cal shape and of the same size are located nearby. We believe that the revealed peculiarities in the state of chromatin (DNA) not only indicate its regular arrangement in the nuclear volume, but, at the same time, provide researchers with a convenient object for a deeper study of the problem of spatial DNA arrange CELL AND TISSUE BIOLOGY
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ment in the interphase nucleus with modern methods of microscopy and molecular biology (Shchapova, 2010). It is suggested that the homologous chromosomes represented by the CG lie along the chromatin helix close to one another. The adjacent arrangement of homologs also seems to be preserved at higher ploidy levels; however, the probability of identical arrange ment of the comparable configurations of three or more consecutive granules on the slide plane possibly decreases (Fig. 12). We are not aware of the mecha nisms of chromosome heterochromatinization and the helical arrangement of heterochromatin granules on the internal nuclear surface in somatic and germ cells upon entry of the silkworm embryo into diapause. DISCUSSION The close correspondence revealed in this work between ploidy of the cell of diapausing embryo and the number of CG in its nucleus (Figs. 1 and 2) allows studying the polyploidization processes that seem to begin as early as at the cleavage period and stop together with mitotic divisions when the embryo enters into diapause. The obtained data show that 4n cells appear at the studied period of development sig nificantly more often in parthenogenetic than in zygotic development; moreover, in the first case, cells of higher ploidy levels—6n and 8n—are also found in the embryo. Whereas the appearance of octoploid cells is easily explainable by two consecutive endomitotic cycles, the appearance of 6n cells requires a different mechanism. We believe that such an additional mechanism specific to parthenogenesis is fusion of nuclei in the cleavage process, which seems to be caused by the same factors
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gc gc
sc gc
(b) gc
sc
gc (a)
(c)
Fig. 9. Germ cells (gc) among somatic cells (sc) in parthenogenetic embryo of P29 clone. (b) germ cells differ by nuclear size; (c) “loose” chromatin granules in gc, the number of which can be estimated by comparing with chromatin of somatic 2ncells. Obj.: 40× (a) and 100× (b, c).
Number of cells, % 100 80 60 40 20 0 40
50
60
70
80 90 100 110 Number of chromatin grains per nucleus
Fig. 10. Distribution of germ cells (gc) of parthenogenetic embryos on the amount of decompacted granules of chromatin CG per nucleus (N = 60). CELL AND TISSUE BIOLOGY
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289 sc
gc
gc sc
sc gc sc (a)
(b)
(c)
Fig. 11. Germ cells (gc) among somatic cells (sc) in zygotic embryo of the re971 line at obj. 20× (a) and 100× (b, c). gc are distinguished by the sizes of the nucleus and cytoplasm, finely disperse chromatin, and huge nucleoli; nuclei of sc are easily detected by compact chromatin and round nucleoli of the same volume.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 12. Helical disposition of chromatin granules in nuclei of diapausing embryo of clone P29 on consecutive optical sections (a–h).
that, in normal (zygotic) development, provide karyo gamy. The karyogamy mechanism in the cytoplasm of the mature unfertilized egg is preserved in thermal par thenogenesis, is added to the process of cleavage of the diploid female pronucleus (Klymenko, 2001), and leads with a certain probability first to fusion of diploid cleavage nuclei and then to secondary nuclear combi nations. Through the fusion of nuclei, 6n cells can appear via two pathways: via triple fusion 2n + 2n + 2n CELL AND TISSUE BIOLOGY
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or via secondary fusion 2n + (2n + 2n); additional tet raploid and octaploid cells of the parthenogenetic embryo appear in a similar way. The mechanism of karyogamy during parthenogenesis is confirmed by the fact that almost half of hexaploid cells (Table 3) form in the head part of the embryo, i.e., in the region of the cytoplasm in which karyogamy usually occurs (Taz ima, 1978).
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(a)
(b)
(c)
Fig. 13. Spiral disposition of chromatin granules in diapause at diploid (a), triploid (b) and tetraploid levels (c).
It cannot be ruled out that part of the appearing polyploid nuclei occurs over time in the area of forma tion of germ cells and can lead to the appearance of polyploid eggs in the grain of parthenogenetic clones. Indeed, tetraploid eggs were found in diploid clones soon after discovery of parthenocloning (Astaurov, 1940), while hexaploid oocytes were detected in trip loid clones upon production of bisexual tetraploid silkworms (Astaurov, 1968). In the present work, poly ploidization of PGCs of diploid clones was directly confirmed (Fig. 10); however, detection of 6n cells in the germ line of diploid parthenoclones needs a signif icantly higher number of embryos in diapause. In any case, the differences of polyploidization processes in the parthenogenetic and zygotic variants of the predi apause development are, in our opinion, rather signif icant and farreaching if we extrapolate polyploidiza tion through fusion of nuclei to natural parthenogene sis (Klymenko et al., 2011). Another aspect in which the two considered vari ants of development differ essentially is chromatin morphology in germ and somatic embryo cells (Figs. 9 and 11). First, we found the more active state of genetic material in the germ line as compared with somatic cells (based on the expressed chromatin decompactization) to be an unexpected and intriguing fact. Second, it was obvious that chromosome decom pactization in the case of fertilization progresses sig nificantly further than in the case of parthenogenesis. The physiological significance of the more active state of the germ line cells in the embryo during diapause in both cases remains unclear. In both types of development, in embryonic somatic cells, the disposition of CG one after another is arranged in such a way that they form a helix on the internal nuclear surface (Figs. 12 and 13). Finer stud ies by modern methods are needed to find whether the adjacent CG are connected to something or occupy predetermined positions in the nuclear envelope architectonics. Based on our microscopic study, we are inclined to suggest that homologous granules, i.e., heterochromatin clumps of identical shape, are located nearby even in polyploid forms (Fig. 13). Does
only the right or only the leftcoiled helix occur here, or are both helices present? How is the spirality of the diapause chromatin connected with the arrangement of the chromosomes in the metaphase plates of mitosis and meiosis, with the spatial distribution of chromo somal DNA in actively working interphase diploid and polyploid nuclei (the silk gland), as well as in diapaus ing nuclei of polyploid extraembryonic cells, where, due to the chromatin fine granularity, ploidy has not yet been established (Figs. 6 and 7). Whereas DNA in somatic nuclei in diapause forms a helix on which homologous granules seeming to make up part of homologous chromosomes are arranged next to each other and follow each other in a tandem, homologous chromosomes in metaphase of meiosis I both of diploids and of tetraploids are arranged in pairs on the “band” of synaptonemal com plex one opposite the other (Klymenko and Spiri donova, 1979). The DNA and chromatin organization in the cell nuclei of the soma and the germ line can differ essentially at the same stages of development (Figs. 9 and 11). However, in the germ line, as in somatic cells, there mitosis occurs and, if it is preceded by adjacent chromosomes, this position can also be preserved in the mitotic metaphase plate, what can be checked by cytological methods. Transition to meiotic DNA orga nization seems to occur in the interphase preceding the meiotic prophase; in the meiosis II metaphase, two homologues are presented only in silkworm tetraploids (Astaurov, 1968) and their mutual arrangement also is available for analysis (Shchapova, 2010). The performed study also has applied significance, as it includes a rather simple method for determina tion of ploidy in any diapausing offspring, that is obtained in experimental genetics and selection when various methods of grain treatment are used to induce polyploidy (Klymenko and Spiridonova, 1982). The experimental material after enter diapause can be ana lyzed without hurry by the method presented in this work and can give reliable estimation of the efficiency of the technique used for obtaining polyploids in the silkworm. CELL AND TISSUE BIOLOGY
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0
0
3
2
3
1
3
26
44.8
2.0
7
8
9
10
11
12
13
Σ
Portion, %
Mean
0
5
1
1
4
6
4
3
2
3
5
6n
1
Embryo
2012
0.5
26.1
6
0
0
0
0
0
0
0
0
0
0
2
3
1
8n
a
0.0
0.0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
PGC
0.6
13.8
8
0
5
0
0
0
1
0
0
0
1
0
1
0
6n
0.1
4.4
1
0
0
0
0
0
0
0
0
0
0
0
1
0
8n
b
0.2
2.9
2
0
0
0
0
0
2
0
0
0
0
0
0
0
PGC
0.2
5.2
3
0
1
1
0
0
0
0
1
0
0
0
0
0
6n
0.4
21.7
5
0
2
1
0
0
0
0
0
0
0
0
1
1
8n
c
2.2
42.7
29
0
4
8
0
4
0
0
6
0
0
5
2
0
PGC
Parts of embryo
0.7
15.5
9
0
0
1
0
0
2
0
1
0
2
0
3
0
6n
0.2
13.0
3
0
0
0
0
0
0
0
0
0
1
0
2
0
8n
d
2.4
45.6
31
5
2
8
0
0
2
0
0
7
6
0
1
0
PGC
0.9
20.7
12
0
1
3
0
0
6
0
0
0
0
0
0
2
6n
0.6
34.8
8
2
6
0
0
0
0
0
0
0
0
0
0
0
8n
e
0.5
8.8
6
0
0
0
0
0
0
0
0
0
0
0
0
6
PGC
Table 3. Distribution of polyploid (6n, 8n) and primordial germ cells (PGCs) along the body of diapausing parthenogenetic embryo (13 embryos of parthenoclone P29) divided into five approximately equal parts (a–e)
CHROMATIN IN DIAPAUSE OF THE SILKWORM 291
292
KLYMENKO, LIANG HAOYOUAN
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CELL AND TISSUE BIOLOGY
Vol. 6
No. 3
2012
