Chromosoma (Berl.) 79, 215 224 (1980) CHROMOSOMA �9 by Springer-Verlag 1980

Chromatin Organization During Meiotic Prophase of Bombyx mori

J.B. Rattner 1, M. Goldsmith 2, and B.A. Hamkalo 1, 2

1 Department of Molecular Biology and Biochemistry and 2 Department of Developmental and Cell Biology, University of California at Irvine, Irvine, California 92717, U.S.A.

Abstract. Chromatin organization during the early stages of male meiotic prophase in Bombyx mori was investigated by electron microscopy. The analysis of nuclei prepared by the Miller spreading procedure, suggests that chromatin fibers which are 200 300 A in diameter undergo an orderly tolding coincident with the formation of the synaptonemal complex. In very early stages the chromatin is released in linear arrays typical of interphase chroma- tin material. With time loops containing 5 25 g of B conformation DNA, initially visualized at the periphery of early meiotic prophasc nuclei, aggregate into discrete tbci. These foci coalesce to form the longitudinal axis of the chromosome in conjunction with the initial appearance of the axial elements of the synaptonemal complex. At pachytene, the loops are evenly distributed along the length of the chromosome and extend radially so that in well spread preparations the chromosome has a brush-like appearance. Through- out this period nascent RNP-fibers were visualized along some of the loops.

Introduction

The elucidation of the particulate structure of eukaryotic chromatin has provided a new basis for the understanding of the organization of eukaryotic chromo- somes. The basic chromatin fiber is composed of a tandem array of nucleosomes that appears to be folded in vivo to form a higher order fiber with a diameter 200-300N (Finch and Klug, 1976; Ris and Korcnberg, 1978; Rattner and Hamkalo, 1978a, b). Several models based on the coiling or folding of this fiber have been proposed to explain the manner in which higher ordcr chromatin structures (e.g., chromosomes) are formed (Dupraw, 1970; Sedat and Manueli- dis, 1977). In contrast, recent biochemical and electron microscopic investiga- tions of metaphase chromosomes depleted of histones and some non-histone proteins by high salt or by competition with the polyanions dextran sulfate and heprin have suggested that the 200-300 ~ fiber is arranged into a series of loops which extend radially from a central element or scaffold which extends

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216 J.B. Rattner et al.

along the axis of the chromatid (Adolph et al., 1977). Support for this arrange- ment has come from thin sections of isolated metaphase chromosomes in which loops of chromatin fibers are seen radiating from the less dispersed central region of the chromatid (Marsden and Laemmli, 1979; Adolph, 1980). A similar arrangement of loops have been observed extending from the margin of meta- phase chromosomes prepared by the Miller spreading procedure although a scaffold structure was not visualized (Rattner and Hamkalo, 1978 a).

Although evidence for the existence of a scaffold structure as a discrete component of metaphase chromosomes in vivo remains inconclusive, it is well documented that in most eukaryotes chromatin is associated with a protein element, the synaptonemal complex during meiotic prophase. This structure forms in association with the pairing of homologous chromosomes and appears to play a role in crossing over and chiasma formation (Moses, 1968).

The arrangement and composition of chromatin fibers during meiosis has not received the same attention as comparable studies on mitotic material. Whole mount preparations of male meiotic pachytene chromosomes suggest that chromatin fibers extend perpendicular to the lateral elements of the synap- tonemal complex and form loops reflecting a lampbrush type organization (Com- ings and Okada, 1970, 1971). In addition, R N A synthesis has been demonstrated in association with these chromatin loops by electron microscopy and autoradi- ography of mouse pachytene chromosomes (Kierszenbaum and Tres, 1974). In the present study, we present observations on the early stages in male meiotic prophase nuclei of the silkmoth Bombyx mori prepared for electron microscopy by the Miller spreading procedure. This procedure allows a more detailed analy- sis of chromatin organization than has previously been reported.

Materials and Methods

Silkworm hybrids of strains C108, 73, and Ascoli were reared on artificial diet (Takeda Chemical Co., Tokyo, Japan) in plastic petri dishes. Diet powder was rehydrated with 1.25-1.5 volumes of distilled water, pressed into thin cakes between sheets of glassine weighing paper, and autoclaved in glass petri dishes for 5 min. Larvae were given fresh diet powder daily and cleaned every other day. Larvae developed 2 3 times slower on diet powder than on fresh mulberry leaves.

Electron Microscopy

The testis was removed from either a 3 or 4th instar Bombyx mori larva and transferred to a solution containing either 1% NP40 (pH 8.5) or a solution containing a three-fold dilution of Joklik's suspension medium with 1% NP40 detergent (pH 8.5). The testis was teased apart with two jewelers forceps and allowed to disperse for 5-10 minutes, and then 0.2 ml of the sample was transferred to a centrifugation chamber containing a carbon coated electron microscope grid and a cushion of 1 M sucrose (pH 8.5). The specimens were processed for electron microscopy by the procedure of Miller et al. (Miller and Beatty, 1969; Miller and Bakken, 1972) and stained with ethanolic uranyl acetate.

Results

Chromatin from meiotic prophase nuclei display several morphologies that are dependent upon the preparative conditions. Higher order fibers (200-300

Fig. 1. Margin of early meiotic prophase nucleus in which loops of chromatin can be identified (arrow). The dense black material is cellular debris. Bar=0.5 gm

Fig. 2. Early meiotic prophase nucleus displaying prominent chromatin fiber loops which are arranged in a linear array and frequently aggregated into discrete foci (arrows). Bar = 1 gm

Figs. 3 and 4. Meiotic chromosomes in which axial elements of the SC can be identified. The chromatin loops extend radially from these elements and appear homogenously distributed along the length of the chromosome. Bar 1 gm

Meiotic Chromosome Structure in Bombyx 219

Fig. 5. A minimal by dispersed chromosome illustrating the condensation of the chromatin loops along the SC. Bar = 1 gin

in diameter) are best preserved when cells are lysed in the presence of media containing divalent cations and 1% NP40. NP40 alone results in a partial relaxation of the fiber and provides the most suitable preparations for the anaylsis of chromosome organization. The addition of 10% formalin (pH 8.5) to the sucrose cushion results in a relaxation of chromatin fiber organization to a beads-on-a-string conformation.

Analysis of electron micrographs of samples prepared from different stages in early meiotic prophase suggests that higher order chromatin fibers undergo an orderly folding coincident with the formation of the synaptonemal complex (SC). Initially, early meiotic prophase nuclei display chromatin fibers which typically show no apparent organization above the 200-300 ~ fiber. Occasional- ly, however, loops can be identified in chromatin at the periphery of spread nuclear material (Fig. 1). As prophase chromatin condensation proceeds, spread preparations display numerous chromatin loops which appear to be arranged first into loose linear arrays; these coalesce at various points to form discrete foci often containing electron dense centers. The association of adjacent foci results in the definition of the longitudinal axis of the meiotic chromosome and the appearance of the axial elements of the SC (Fig. 2). At the completion of this process chromatin fibers appear as a series of radially arranged loops which are evenly distributed along the completely formed axial elements of

220 J.B. Rattner et al.

Fig. 6. Meiotic chromosome displaying prominent RNP fibrils (arrows) associated with chromatin loops extending from various points along the length of the chromosome. Bar=0.5 gm

the SC (Figs. 3, 4). This arrangement is maintained during the pairing process and the formation of the mature SC so that at pachytene the loops radiate from the SC imparting a brush-like appearance to the chromosome. This ex- tended brush-like configuration results from some degree of relaxation of the loops due to the preparative procedure and probably does not reflect the more compact organization of the chromatin fibers in vivo. Rather, additional folding of the loops most probably results in the folding of chromatin in close apposition with the SC. This compact configuration is suggested by chromosome prepara- tions that have been allowed the minimum of dispersal time (Fig. 5).

Because of the variation in the degree of chromatin fiber dispersal in these preparations it is difficult to precisely determine the length of the chromatin loops. In addition, the organization of the nucleosomes within the higher order fiber remains undefined. A packing ratio of 1:21 to 1:40 has been estimated for the D N A based on either a "superbead" or helical folding of the nucleosome containing fiber within the higher order fiber (see Hozier 1979 for review). Based on these ratios we estimate that the loops present on our preparations contain 5-25 lam of B DNA. The distribution of the loop sizes appears to be the same throughout the meiotic prophase stages investigated as well as for loops that are either transcriptionally active or inactive.

Figs. 7 and 8. Transcriptionally active chromatin from the chromatin loops of early meiotic chromo- somes. Ba r=0 .5 ~tm

Fig. 9. Meiotic prophase chromosome displaying highly transcriptionally active chromatin loops (arrow). Note the absence of a discrete axial element in the region of active transcription. Bar = 1 gm

222 J.B. Rattner et al.

A majority of the chromatin loops appear to be transcriptionally inactive along the length of the chromosome. There are, however, loops displaying ribonucleoprotein fibers along their lengths (Fig. 6). The RNP fibers were distin- guished as more densely-staining fibers extending from the main chromatin axis and, in dispersed preparations, nucleosomes were seen along the fiber connecting adjacent RNP fibers. Most active chromatin loops displayed a low fiber frequency and do not show well defined fibril gradients; this morphology is typical of non-ribosomal gene transcription in many systems (McKnight and Miller, 1976; Foe et al., 1976; Laird et al., 1976). Occasionally, well-defined fibril gradients such as that illustrated in Figures 7 and 8 were observed. Loops displaying a high rate of transcriptional activity appear in less dispersed prepara- tions to be coated with RNP fibrils along the entire length of the loop; frequently the SC is disrupted in these regions (Fig. 9). The fact that RNP fibrils were seen attached to the chromatin throughout the phases of chromatin condensation suggest that active non-ribosomal RNA synthesis is occurring throughout early meiotic prophase.

Discussion

Electron microscope examination of early meiotic prophase nuclei clearly dem- onstrates the orderly packing of chromatin fibers into discrete loops. These preparations suggest that, at least at the initial stages, this folding occurs indepen- dent of the formation of the central components of the SC although loop aggregation is associated with the appearance of the axial elements as prophase continues. The absence of a discrete SC in regions of high transcriptional activity may suggest that this structure is not necessary for the maintenance of the loop organization. The loop-like organization of meiotic chromatin was pre- viously detected in water spread preparations of pachytene nuclei (Comings and Okada, 1970, 1971); a similar conformation has also been observed in whole mount and thin sections of isolated mitotic metaphase chromosomes (Adolph et al., 1977; Marsden and Laemmli, 1979; Adolph, 1980). Thus, the arrangement of the chromatin loops observed in meiotic prophase may represent a universal mode of folding of the 200-300 N chromatin fibers into even higher order structures. It is possible that the organization of the 200-300 ~ chromatin fiber into loops, and subsequently into chromosomes, is determined by the distribution of non-histone chromosomal proteins and their interaction with specific DNA sequences. The central regions of the foci may contain those proteins which are responsible for chromatin fiber packing at both meiosis and mitosis. Such an arrangement may account for the localization of "scaffold- ing proteins" along the longitudinal axis of mitotic chromosomes (Adolph et al., 1977). At meiosis additional proteins unique to the SC would be localized in this region. At the present time we do not know if there is a relationship between loop-size and chromosome organization. However, it is interesting to note that the estimated loop sizes (5-25 gm band of B DNA) of meiotic prophase

Meiotic Chromosome Structure in Bombyx 223

c h r o m o s o m e s are s imi lar to the values ob ta ined for loops in H e L a cell me taphase c h r o m o s o m e s ( A d o l p h et al., 1972).

I t has p rev ious ly been suggested tha t the d i s t r ibu t ion of the loops a long the SC is respons ib le for the f o r m a t i o n o f ch romomeres seen in the l ight micro- scope (Comings and O k a d a , 1970). Our p repa ra t i ons suggest tha t the loops are evenly d i s t r ibu ted a long the SC; thus var ia t ions in fo ld ing above the level of the loops m a y be respons ib le for c h r o m o m e r e organiza t ion .

The v isua l iza t ion of nascent RNP- f ib r i l s on the ch roma t in loops is in agree- men t wi th previous repor t s o f mouse meio t ic p r o p h a s e c h r o m o s o m e s (Kierszen- b a u m and Tres, 1974). A ma jo r i t y of t ranscr ip ts i l lus t ra ted in our p r e pa ra t i ons d i sp lay a pa t t e rn which is typical of n o n - r i b o s o m a l t r ansc r ip t ion and our da t a suggests tha t R N A synthesis is occur r ing t h r o u g h o u t early meio t ic prophase . These observa t ions are consis tent wi th ear l ier studies of R N A synthesis dur ing meio t ic p r o p h a s e of insects (Henderson , 1964; Das et al., 1965).

Acknowledgement. The authors would like to thank Nancy Hutchison for very helpful comments during the preparation of this manuscript. This work was supported by National Science Foundation grant PCM 78-08930 to B.A.H. and J.B.R., and N.I.H GM27302 to M.G. Dr. Hamkalo is a recipient of Research Career Development Award GM00233.

References

Adolph, K.W. : Isolation and structural organization of human mitotic chromosomes. Chromosoma (Berl.) 76, 23-33 (1980)

Adolph, K.W., Cheng, S.M., Laemmli, U.K.: Role of non-historic proteins in metaphase chromo- some structure. Cell 12, 80~816 (1977)

Comings, D.E., Okada, T.A.: Whole mount electron microscopy of meiotic chromosomes and synaptonemal complex. Chromosoma (Berl.) 30, 269-286 (i970)

Comings, D.E., Okada, T.A.: Whole mount electron microscopy of human meiotic chromosomes. Exp. Cell Res. 65, 99-103 (1971)

Das, W.K., Siegel, E.P., Alfert, M.: Synthetic during spermatogenesis in the locust. J. Cell Biol. 25, 387-395 (1965)

Dupraw, E.J.: DNA and chromosomes. New York-Holt: Rinehart and Winston 1970 Finch, J.T., Klug, A.: Solinoidal model for superstructure in chromatin. Proc. nat. Acad. Sci.

(Wash.) 73, 1897-1901 (1976) Foe V.E., Wilkinson, L.E., Laird, C.D.: Comparative organization of active transcription units

in Oncopeltus fasciatus. Cell 9, 131-134 (1976) Henderson, S.A.: RNA Synthesis during male meiosis and spermogenesis. Chromosoma (Berl.)

15, 345 366 (1974) Hozier, J.C. : In: Molecular genetics, part III. Chromosome structure (J.H. Taylor, ed.), pp. 315-380.

New York: Academic Press 1979 Kierszenbaum, A.L., Tres, L.L. : Transcription sites in spread meiotic prophase chromosomes from

mouse spermatocytes. J. Cell Biol. 63, 923-925 (1974) Marsden, M.P.F., Laemmli, U.K.: Metaphase chromosome structure: evidence for a radial loop

model. Cell 17, 84%858 (1979) McKnight, S.L., Miller, O.L., Jr. : Ultrastructural patterns of RNA synthesis during early embryo-

genesis of Drosophila melanogaster. Cell 8, 305-319 (1976) Miller, O.L., Jr., Bakken, A.H. : Morphological studies of transcription. Acta endocr. (Copenhagen)

168, 155-177 (i972)

224 J.B. Rattner et al.

Miller, O.L., Jr., Beatty, B.R. : Visualization of nuclear genes. Science 164, 955-957 (1969) Moses, M.J.: Synaptinemal complex. Ann. Rev. Genet. 2, 363-412 (1968) Rattner, J.B., Hamkalo, B.H.: Higher order structure of metaphase chromosomes. I. The 250

fiber. Chromosoma (Berl.) 69, 363-372 (1978 a) Rattner, B., Hamkalo, B.A. : Higher order structure of metaphase chromosomes. II. The relationship

between the 250 A fiber, superbeads and beads-on-a-string. Chromosoma (Berl.) 67, 373-379 (1978b)

Ris, H., Korenberg, J.: Chromosoma structure. In: Cell biology (L. Goldstein and N. Prescott, eds.), Vol. II. New York: Academic Press 1978

Sedat, J., Manuelidis, L.: A direct approach to the structure of eukaryotic chromosomes. Cold Spr. Harb. Syrup. quant. Biol. 42, 331-350 (1977)

Received March 17, 1980 / Accepted March 25, 1980 by R.B. Nicklas Ready for press April 2, 1980