THE AMERICAN JOURNATJ OF ANATOMY 189:11-23 (1990)

Three Structures Associated With the Nucleolus in Male Rat Germinal Cells: Round Body, Coiled Body, and “Nubecula” and General Presence of Round Body at Male Meiosis MICHAEL C . SCHULTL Department of Anatomy, McGill University, Montreal, Quebec, Canada, H3A 2B2

In addition to chromosomes and ABSTRACT nucleoli, three structures, i.e., round body, coiled body, and nubecula, are encountered in the nucleus during the meiotic prophase in male rats. These structures have been examined by electron microscopy in random and serial sections. The round body is a finely fibrillar, proteinaceous structure closely associated with the granular component of a nucleolus in rat spermatocytes and young spermatids. A similar structure has been observed in man, the monkey Macaca mulatta, the gastropod Achatina fulica, and the insect Locusta migratoria. Together with evidence from the literature, these results support the view that the round body is of general occurrence in the male meiocytes of eukaryotes and may, therefore, play a role in meiosis. The coiled body is a group of electron-dense elements called “coils”, which average 35 nm in width, except after mid-pachytene when their size almost doubles. The coils are composed of 2-nmwide filaments and 8 to 10-nm-widegranules, both of which are ribonucleoprotein. The coiled bodies are interpreted to be groups of “spliceosomes”, that is, structures containing heterogeneous RNA and small nuclear RNA. A remarkable feature of the coiled body is its temporary disappearance at early pachytene and its reappearance at late pachytene, possibly due to drastic changes in the turnover rate of its component RNAs. The nubecula is a newly identified nuclear inclusion, composed of weakly staining threads loosely organized into a 560nm-wide spheroid. It has been observed only in early pachytene nuclei. INTRODUCTION

While rat nucleoli undergo extensive changes during and after meiosis, as described in the companion article (Schultz and Leblond, 1990), three other structures are usually associated with them. One, the round body, previously studied in the rat (Schultz et al., 1984),has been identified in association with meiotic nucleoli in man and monkey a s well as in two invertebrates, the gastropod Achatina fulica and the insect Locusta migratoria. The second structure is a collection of electron-opaque elements identical to the coiled body previously described in the somatic cells of plants (Moreno Diaz de la Espina et al., 1982) and animals (Bernhard, (c)

1990 WILEY-I,ISS, INC.

1971). Because to our knowledge the existence of a coiled body has not been reported in spermatogenic cells, this structure will bc described in primary spermatocytes as they undergo the long meiotic prophase. Thirdly, a nuclear structure that has not been previously reported in any cell type will be described in rat primary spermatocytes. Because of its size, shape, and fluffy appearance, this new inclusion has been named nubecula (from Latin, “little cloud”). MATERIALS AND METHODS

In general, the rat testis was fixed by perfusion with 5%)glutaraldehyde in 0.1 M sodium cacodylate, pH 7.3, and postfixed by the reduced osmium method of Karnovsky (details in Schultz et al., 1984). After embedding in Epon, random and serial sections were prepared a s indicated in the accompanying article (Schultz and Leblond, 1990). Demonstration of a Round Body in Primary Sperrnatocytes of Man, Monkey, Snail, and Locust

Male germinal tissues were fixed and embedded in Epon for electron microscopic examination a s indicated in Table 1. Human testes from a fertile adult male given therapeutic bilateral orchiectomy were kindly made available by Dr. M. Leplant (Montreal General Hospital). Epon-embedded samples of testicular tissue from a n adult monkey (Hermo et al., 1977) were provided by Dr. L. Hermo (Department of Anatomy, McGill University). Invertebrate specimens raised in the laboratory (gifts from Drs. R.W. Chase and M. Robertson, Department of Biology, McGill University) were dissected under the primary fixative; and prophase I spermatocytes, identified by the presence of synaptonemal complexes in the nucleus, were examined in cysts of the gonad that included late spermatids with apparently normal, condensed nuclei. Tissue sections were stained according to the usual uranyl-lead procedure. Demonstration of Coiled Body and “Nubecula” in Rat Spermatogenic Cells

These structures were examined in serial electronmicroscopic sections from which data on the nucleolus

Received October 29, 1988. Accepted March 26, 1990. Address reprint requests lu Dr. C. P. Leblond, Department of Anatomy, McGill University, Montreal, Quebec, Canada H3A 2B2. Dr. M.C. Schultz’s present address is Fred Hutchinson Cancer Research Center, Seattle, Washington, 98104.

12

M.C. SCHULTZ

TABLE 1. Summary of the fixation protocols used to prepare male germinal tissues from animal species surveyed for the presence of a round body SDecies Man (Homo sapiens) Monkey (Macaca mulattal Snail (Achatina fulicu) Locust (Locusta migratoriai

Gonad dissected in: Medium 199, modified Primary fixative Primary fixative Primary fixative

Primary fixation 5% glutaraldehyde, immersion 2 hr 5%glutaraldehyde, perfusion 30 min 1%glutaraldehyde + 1%formaldehyde, immersion 2 hr 4%)glutaraldehyde, immersion 2 hr

were also obtained (Schultz and Leblond, 1990). In addition, a histochemical study of the coiled body was carried out, using phosphotungstic acid for the detection of protein by the method of Silverman and Glick (1969), and using ribonuclease-bound gold for the detection of RNA by the method of Bendayan (1981a,b). Finally, the response of the coiled body to silver impregnation was examined according to the methods of Williams et al. (1982) modified as follows. Primary fixation was by perfusion with 2.5%glutaraldehyde in 0.1 M Sorensen’s phosphate buffer containing 4% sucrose, pH 7.4, and subsequent immersion in the same solution for 2 hr. The testis was then cut by hand into thick slices, which were mounted in agar and sectioned at 100 Frn with a tissue chopper. The sections were then left for 10 min in Carnoy’s solution, decerated, impregnated with 50% silver nitrate for 25 h r at 50°C in the dark, rinsed in distilled water at 50”C, and developed w/v in a 1:I mixture of 80% silver nitrate and 1.1%) formaldehyde, pH 5.3, for 20 min a t 50°C. The material was then processed for Epon embedding. Thin sections were examined after staining for 2.5 min with 4% uranyl acetate in water. RESULTS The Round Body

Although the round body of the rat has been described in detail (Schultz et al., 1984), two figures are presented in which its shape and association with nucleolar components are depicted (Figs. 1 , 2 ) for comparison with the results in other species. Homo sapiens (Vertebrata, Mammalia, primates). Primary spermatocytes in the adult human contain a nucleolus-associated body that is spherical and composed of narrow, evenly stained fibrils. It is surrounded by the ribonucleoprotein (rRNP) particles of the granular component, whereas fibrillar centers and the fibrillar component are not in direct contact with it (Fig. 3). Its moderate staining is intermediate between that of fibrillar centers and the fibrillar component, as in the rat round body (Fig. 11, and it sometimes displays electron-lucent lacunae, again a s in the rat (Fig. 2). Hence the structure is similar to the r a t round body, particularly as it appears in diplotene spermatocytes (Fig. 2). It is therefore taken to be a “human round body.” Macaca mulatta (Vertebrata, Mammalia, primates). The nucleolar granular component in primary spermatocytes of the monkey, M . rnulatta, contains a struc-

Primary fixative vehicle and wash buffer 0.1M cacodylate, pH 7.4 0.2M s-collidine, pH 7.2

0.05M Sorensen’s phosphate buffer, pH 7.4 0.1M cacodylate, pH 7.4

Secondary fixation Reduced osmium tetroxide, 1 hr 1%osmium tetroxide, 1-2 hr Reduced osmium tetroxide, 1 h r Reduced osmium tetroxide, 1 hr

ture (Fig. 4) which is spherical, is predominantly formed of fine fibrils, and sometimes displays a prominent, electron-lucent lacuna. This structure is similar to the rat round body after mid-pachytene, except that the component fibrils intermingle with more strongly stained rRNP particles of the same size (15-20 nm) as those of the surrounding granular component. With this exception, the structure is similar in shape and staining properties to the r a t round body and is, therefore, considered a “monkey round body.” Achatina fulica (Mollusca, Gastropoda, Pulmonata). The nucleoli of primary spermatocytes in the snail, A. fulica, contain a spherical structure formed of narrow fibrils with moderate electron opacity, in which there is occasionally a small lacuna (Fig. 5). Although distinct from fibrillar centers, it is surrounded by granulofibrillar nucleolar material. Except for the presence of granulo-fibrillar instead of plain granular component around it, this structure is similar to the rat round body. Locusta migratoria (Arthropoda, Insecta, Orthoptera). The nuclei of primary spermatocytes in the locust, L. migratoria, contain two types of spherical structures composed of narrow filaments and associated with the nucleolus (Fig. 6). One of these appears pale and occurs in several copies per nucleus. It is similar to, and probably homologous to, fibrillar centers in mid pachytene r a t spermatocytes (Fig. 1).The staining of the other spherical structure is intermediate between that of the putative fibrillar center and the nucleolar cords. Only one copy of this moderately stained structure is ever encountered in a nuclear profile. Owing to its composition, staining, and frequency, the moderately electron-opaque structure is considered to be a homologue of the round body in r a t primary spermatocyte. The cords of the nucleolus shown in Figure 6

A hhrruiatioris C

CB Chr f fc g

L NE RB

coils coiled body

heterochromatin fibrillar component fibrillar center granular component lacuna of round body nuclear envelope round body

Fig. 1. Rattzrs noruegicus. Nucleolus of a rat primary spermatocyte at mid-pachytene. A pale and finely textured fibrillar center is conlinuous with cnrds of‘the mom opaque fibrillar component. The granular component extends away from the fibrillar center to envelop the round body, whose staining intensity is intermediate between that of the fibrillar center and fibrillar component. x 29,500.

Fig. 2. Nucleolus of a rat primary spermatocyte at diplotene, showing a condensed fibrillar center, fairly compact fibrillar component, and granular component that surrounds the round body. x 40,700.

for L. migratoria are formed either of fibrils or of a n admixture of granules and fibrils. These cords approach the surface of the locust round body, but do not intermingle with its substance. A similar situation is observed in diakinetic spermatocytes of‘ the rat, in which large areas on the surface of the round body are not in contact with rRNP granules (Schultz et al., 1984).

opaque particles that are about half the diameter of the 15-20 nm granules in the granular component (Fig. 7 ) .

The Coded Body Morphological Features

The coiled body of rat spermatogenic cells (Fig. 7) is formed of a group of dense, distinct elements referred to as “coils”, around which are poorly stained spaces continuous with the nucleoplasm and referred to a s “channels” (Fig. 7). The coils include areas of moderate and high electron opacity that often blend gradually. In some sections, the coils show regions of attenuation. The main component of the coils consists of densely packed, undulating filaments that are 2 nrn wide on average (Fig. 7, Table 2). The maximum observed length of these filaments i s 13 nm. In the best sections of coils, it is also possible to distinguish electron-

Development

Type A and B spermatogonia contain a coiled body a t every stage of the cycle ofthe seminiferous epithelium (Schultz, 1986). At pwleptotene and leptotene (stages VII-XI of the cycle), the nucleus contains a single coiled body located close to the granular component of a nucleolus, although there is no intermingling of‘ nucleolar rRNP granules and coils (Fig. 8 ) . In a nucleus examined in serial section at preleptotene (stage VII), the diameter of the coiled body is about 560 nm, the mean width of the coils is 37 nm, and the component filaments measure 2.1 nm across, while the width of the channels is highly variable (Table 2). During early pachytene (stage XIV of the cycle to stage IV of the next cycle), examination of every fifth section in a complete series through two nuclei and of every section in a series through nine-tenths of two other nuclei has shown no coiled body, thus indicating that this structure has disappeared. Toward the end of

14

M.C. SCHU1,'1'%

Fig. 3. Homo supiens. Spherical structure identified as round body within the nucleolus of a human primary spermatocyte. The round body is composed of' fine fibrils and includes a small lacuna. It is surrounded by the granular component along the edge of which is a small fibrillar component in contact with a fibrillar center. x 41,000.

the early pachytene period, that is, a t stage IV (terminology of Leblond and Clermont, 19521, a coiled body reappears (Fig. 9), whose dimensions are similar to those in preleptotene spermatocytes (Table 2). It is usually adjacent to both the granular component and the nuclear envelope. At mid-pachytene, one coiled body is present per nucleus (Table 2). The actual diameter has been found to range between 780 and 820 nm in four serially sectioned, stage VII nuclei, each cut through about threequarters of its volume. The coiled body is again adjacent to the granular component of a nucleolus (Fig. 10) but, as earlier, the peripheral material of its coils rarely intermingles with the rRNP granules of the granular component. A t late pachytene, three nuclei have been examined a t stage XI1 in sets of serial sections that include half to three-quarters of each nucleus, as well a s in four other late pachytene spermatocytes (stage XII) examined in at least every fourth section of a series that includes the entire nucleus. One or two coiled bodies have been observed per nucleus. These coiled bodies have diameters ranaine between 310 and 800 nm. The mean coil width is about twice that at earlier stages Y

Y

Fig. 4.Macuca m.ulnttu. Roughly spherical structure identified as round body in a monkey primary spermatocyte. It is composed of fibrillar material, within which granules are scattered. These granules are of the same size and appearance (arrows)as the rRNP granules making up the surrounding granular component of a nucleolus (which is believed to be a late stage of meiosis). The round body includes a prominent lacuna. (From material provided by L. Hermo.) x 41,300.

(Table 2). A striking change a t late pachytene is a n intimate association of every serially sectioned coiled body with the fibrillar component of a nucleolus (Fig. 11).In one nucleus, two coiled bodies are associated with separate cords of the fibrillar component of the same nucleolus. Coiled body and fibrillar component often associate through apparent intermingling of their cords. On the other hand, the coiled body may also be adjacent to the granular component. Coiled bodies associated with the fibrillar component are present up to the end of diplotene, after which time none are detected in random sections. In particular, no coiled body has been observed in spermatids. Histochemical Properties

The presence of protein in the coiled body was demonstrated by the phosphotungstic acid method. At late pachytene, for example, a strong reaction of the coils (Fig. 12) as well a s of the fibrillar component of a nearby nucleolus was observed, with a weak reaction of the granular comDonent. The search for RNA bv the RNKse-gold method demonstrated moderate" and

EXTRANUCLEOLAR STRIJ( :TI IKP:S I N SPKKMA‘l’Ot‘YTES

15

lucent spaces of variable size and shape (Fig. 18). No other type of material is observed in serially sectioned nubeculae. The nubecula is located next to the nuclear envelope (Figs. 15,161. It is also occasionally located close to condensed chromatin (Fig. 161, the sex pair (not shown), and the nucleolar granular component which may, as in Figure 17, include a round body. DISCUSSION

The significance of the three structures under study-round body, coiled body and nubecula-can to some extent be deduced from their structural features. Round Body

Fig. 5. Achalina fdicu. Spherical structure with minute lacunae (arrow) identified as a round body in a snail primary spermatocyte. Around it, nucleolar material contains highly electron opaque areas of intermingled fibrils and granules as well as separate regions that are predominantly fibrillar (white box) or granular (nucleoplasmic side of white lines). x 41,300.

strong reactions over fibrillar and granular components, respectively; a moderate one over the coiled body; a weak one over chromatin; and none over the round body (Fig. 13). Finally, argentaffinity of the coiled body, as well a s of the fibrillar component, was observed using either silver nitrate impregnation and development according to Williams et al. (1982) as illustrated in Figure 14, or the impregnation method of Knibiehler et al. (1981; not shown). Nubecula

The nubecula is a poorly stained, roughly spherical structure (Figs. 15-18) present only once in each early pachytene (stage I) nucleus examined by serial sectioning. The nubecula in fact is restricted to pachytene nuclei a t stages I-IV (inclusive), since i t has not been seen in serially or randomly cut cells at any other stage. Its actual diameter, the average of the long and short axes at the widest point in stage I serial reconstructions ranges from470 to 730 nm (mean = 560 nm, n = 5). In random sections at stages I1 to IV, the diameter is 490-610 nm. The nubecula is composed of a loose aggregdion of randomly oriented coarse threads that on average are 11.6 nm across (SD = 3.2; n = 30). Individual threads have been observed up to 78 nm long. They stain weakly by comparison with fibrils of chromatin (18.2-nm wide on average, SD 3.4, n = 19) and are separated from one another by electron-

In the rat, the round body was found to be a nucleicacid-free, proteinaceous structure associated with a nucleolus, rarely with two, in spermatocytes and young spermatids (Schultz et al., 1984); i t was not stained by silver impregnation (Schultz, unpublished observations). The present work shows the presence of a similar structure in spermatocytes from two other vertebrate and two invertebrate species (Table 3). The round body that was observed in human primary spermatocytes, like that briefly described by Panigua et al. (19861, closely resembled its homologue in rat. The round body of the snail, Achatzna futica, was also similar to that in the rat, except for smaller lacunae, In the mollkey, Macaca mulatta, the round body differed from that of r a t by its content of granules resembling those in the granular component. A similar condition was reported in the mouse, in which the round body was fibrillar at mid-pachytene but included granules a t late pachytene (Solari, 1969). The round body of the type I nucleolus in the wood lemming (Myopus schisticolor) also contained “dense granules” (Wolf et al., 1987). In Locusta mzgratorra, two fibrillar structures one of low and one of intermediate electron opacity, were associated with the nucleoli of primary spermatocytes. The fibrillar structure with low opacity, presumed to be the one described under the name “spherical body” by Friedlander et al. (19761, was taken to represent a fibrillar center. The other fibrillar structure, characterized by intermediate electron opacity, in the locust resembled the round body of r a t primary spermatocytes a t late prophase, when much of the surface did not contact nucleolar material (Schultz et al., 1984). Examination of the literature on other species in the light of the information on rat, man, monkey, snail, and locust (Table 3) revealed structures that could be interpreted to be homologues of the round body. In particular, pachytene microsporocytes in two plants displayed nuclear inclusions with close resemblance to the round body. First, the structure named “globulus” in onion (Allium cepa; Gimenez-Martin and Stockert, 1970; Stockert et al., 1970)was spherical and formed of narrow filaments, often displayed a prominent lacuna, and was associated with the granular component of a nucleolus. Even though there were up to three of these globuli, they could be homologues of the round body. The second case was observed in corn (Zea mays; Gillies and Hyde, 1973). The structure, referred to a s “dark body”, was also spherical, usually had a prominent lacuna, and was exclusively associated with rRNP-containing elements of the nucleolus. It was

16

Fig. 6. Lomsta migratorza. Spherical structure identified as round body in association with the nucleolus in a locust primary spermatocyte. The round body has a circular outline, i s of moderate electron opacity, and is composed of fine fibrils. Next to it are fibrillo-granular

cords (fg).The nucleolar material also includes a sphere of low density presumed to be a fibrillar center (SB), in association with the fibrillar component. x 40,600.

formed of narrow filaments that reacted intensely to staining of protein with alcoholic phosphotungstic acid. It was again interpreted to be a round body. This reexamination of micrographs published by other workers and the results of a brief survey presented herein (Table 3) lend strong support to the hypothesis that the round body is of general occurrence in male meiocytes. The widespread occurrence of a round body in meiotic cells indicates t h a t this structure has been retained throughout evolution and, therefore, must play some fundamental role in the survival of plant and animal species. Moreover, since it is present in meiotic cells and, a s far was known, not in any other cell (except for a residual one in young spermatids), the round body is likely to exert its function during meiosis. This function possibly involves the production of ribosomes,

since a universal feature of the round body is its association with the nucleolus. Coiled Body The coiled body was first described in somatic cells as a 300-nm- (Monneron and Bernhard, 1969) to 900-nm wide sphere (Seite et al., 1982) composed of units called “threads” (Monneron and Bernhard, 1969; Bernhard, 1971; Sananes and Le Goascogne, 19761, “strands” (Kinderman and LaVelle, 1976; Matsushima et al., 1984) and hereafter “coils” (Kinderman and LaVelle, 1976; Schultz, 1989). The coils had a width varying from 30 nm (Lafarga et al., 1983a1 to 63 nm (Hervas et al., 1980). The coiled body observed in spermatocytes was similar, since its size ranged from 300 to 800 nm and individual coils averaged 35 nm in width up to

17

EXTRANIJCLEOLAR STRUCTIJRER I N SPERMATOCYTES

Fig. 7. Characteristic features of the coiled body as exemplified in a mid-pachytene spermatocyte (Stage VII). It is composed of a number of dense elements referred to as coils separated by channels (asterisks). The channels have the same staining properties as the nucleoplasm and are continuous with it at the periphery of the coiled body (arrow). The coils have intermediate to high electron opacity (between arrowheads middle left) and often present an irregular outline includ-

ing attenuated regions (highlighted by dashed lines). Some coils contain completely electron opaque particles (arrowheads middle right) that are about half the diameter of' granules in the granular component. Inset: High-magnification view of the 2-nm-wide filaments composing the coils (arrowheads). A cross-sectioned filament is arrowed. x 95,800; inset, x 364,000.

TABLE 2. Number of coiled bodies per serially sectioned nucleus and diameter of its components during meiotic prophase in the rat'

Stage of cvcle Preleptotene Early pachytene Early pachytene (end) Mid paehytene Late oachvtene

VII I IV VII XI1

No. of coiled

Approx. size of coiled

bodies p e r

bodies

nucleus'

(nm) 560

1 0

1 1 2

380-450 780-820 310-800

Mean coil width (nm +SD) 37 1 8

Longest coil observed (nd 113

Range o f channel widths (nm) 5-52

Width of component filaments (nm +SD) 2.1 2 0.4

34 k 14 35 i 11 68 t 21

144 136 312

8-40 7-31 9-40

2.5 k 0.4 2.3 t 0.4 2.1 5 0.3

'Coil width measured perpendicularly to the axis of the coils in more than 34 profiles at each period (except a t the end of early pachytene when 17 wore measured). The filaments making up the coils were measured in more than 30 specimens in each case. 'The number of coiled bodies per nucleus was inferred from observations of serial sections, except toward the end of early pachytene when the conclusion was reached from the examination of numerous random sections.

mid-pachytene and 60 nm thereafter. In neurons and other somatic cells (Monneron and Bernhard, 1969; Seite et al., 1982; Lafarga et al., 1983b), the coiled body contained protein and RNA and stained heavily with silver. Similar properties characterized the spermatocyte coiled body. Hence, the appearance as well as the

cytochemical data established that the coiled body in rat spermatogenic cells was homologous to the one in somatic cells. Despite some similarities between the coiled body and the fibrillar component of the nucleolus, they differ in their detailed molecular composition. The fibrillar

18

M.C. S(:HIJLTZ

Fig. 8. Coiled hody in a st.age VII preleptotene spermatoeyte. It is located in the vicinity of the granular component of a nucleolus. Asterisk, channel. x 95,900. Fig. 9. The newly formed coiled body in an early pachytene spermatocyte (stage IV) is located against the inner membrane ofthe nuclear envelolje. As previously, it is formed of cords separated by channels (asterisk). x 94,400.

component is known to contain 45s precursors of rRNA (Goessens, 1984), whereas coiled bodies include heterogeneous (hn) RNA as well as small nuclear (sn) RNA (Fakan et al., 1984). Biochemical studies have established that hnRNA and snRNA’s form the “spliceosomes” in which pre-mRNA splicing occurs (reviewed in Reed et al., 1988). Spliceosomes isolated from HeLa cell nuclei are cordlike structures about 30 nm wide

Fig. 10. Coiled body in a inid-pachytene spermatocyte (stage VI1). It is located against the nuclear envelope and is associated with the reticulated granular component of a nucleolus. Cords of the coiled body may show variable staining intensity (between arrowheads); a t the periphery they are sometimes approached by 15 to 20-nm granules of the granular component (arrows),which, however, do not intermingle with the matcrial comprising the cords. x 95,800.

and up to 60 nm long, except in short regions, where they are attenuated (Reed et al., 1988). They also contain 8 to 10-nm-wide granules. Spliceosomes therefore closely resemble coils. This suggests that coiled bodies could be a site of pre-mRNA splicing in situ and thus be a group of spliceosomes. The grouping of spliceosomes could represent the splicing of pre-mRNA produced a t a given locus and being actively transcribed.

EX'I'ItANUCLEO1,AK STKUCTIJRES I N SPERMATOCYTES

19

Fig. 11. Coiled body in a late pachytene spermatocyte (stage XU). The coiled body includes cords and channels (asterisk),which are continuous with the nucleoplasm at the periphery of the coiled body (large arrow). The coiled body is associated wilh cords of fibrillar component from n nucleolus. The fibrillar coniponent encloses a condensed fibrillar center and intermingles with 15 to 20-nm rKNP granules (small arrows) at its interface with the granular component. x 95,800.

In somatic cells, investigators presumed that the coiled body was a permanent component, except Lafarga et al. (1983a) who examined random sections and described its disappearance during the postnatal differentiation of immature glial cells. Here, the results from serial sectioning suggested the disappearance of the coiled body toward the end of leptotene (stage XI) and its absence during zygotene (stages XI1 and XIII) and most of early pachytene (stage XIV and stages 1-111 of the next cycle), that is for a period of 5.3 days. Toward the end of early pachytene (stage IV), the coiled body reappeared. It then persisted until the first metaphase of meiosis. In view of the proposal that coils contain pre-mRNA being spliced, the disappearance of the coiled body would represent the completion of splicing,

while its reappearance a t stage IV would indicate resumption of splicing. Nubecula

The nubecula was a poorly stained structure composed of thick, irregular threads present within uniform material forming its background. To our knowledge, this inclusion had not been described in spermatogenic cells. Gross resemblance to the "spherical body" of Friedlander et al. (1976) in locust spermatocytes (Fig. 6, cf. nubecula in Fig. 16) was apparent, although several importance features distinguished these structures: (1)in random sections, the nubecula was small and rare, whereas the larger spherical body was common; (2) only one nubecula was

20

M.C. SCHIJLTZ

Fig. 12. Response of the coiled body in a late pachytene spermatocyte (stage XII) to reaction with phosphotungstic acid for the detection of protein. Like the fibrillar component of the nearby nucleolus, the coils of the coiled body are heavily stained. Its channels (for example, between the cords labeled c), as well as the nucleoplasm are little or not stained. x 54,400.

Fig. 13. Ribonuclease-gold labeling of the coiled body in a late pachytene spermatocyte (stage XII).The condensed granular component is heavily labeled, and a number of gold particles lie over the fibrillar component and the coils of the coiled body, while the round body is completely unlabeled. x 54,400.

21

EXTRANUCLEOLAR STRUCTURES IN SPERMATOCYTES

Flg. 14. Distribution of silver deposits in the nucleus of a late pachytene spermatocyte (stage XII) after impregnation with 50%silver nitrate. Heavy staining is observed in the coils (arrow) of the coiled body. The fibrillar component is also associated with heavy

deposits of silver. Heterochromatin blocks are stained most heavily a t their periphery. Arrowheads indicate the position of the nuclear envelope. ~ 3 2 , 1 0 0 .

TABLE 3. Summary of data available concerning the phylogenetic distribution of the round body in male gametocytes Species Primates Human Macaca mulatta Lower mammals Rat Mouse Chinese hamster Ph,yllotis darwini Myopus schisticolor Microtus agrestis Invertebrates Achatina fubica Locusta rnigratoria Plants A l l i u m cepa Zea mays

Phylum, class, order

Evidence for round body’

Vertebrata, Mammalia, Primates Vertebrata, Mammalia, Primates

This paper This paper

Vertebrata, Mammalia, Rodentia Vertebrata, Mammalia, Rodentia Vertebrata, Mammalia, Rodentia Vertebrata, Mammalia, Rodentia Vertebrata, Mammalia, Rodentia Vertebrata, Mammalia, Rodentia

This paper Previous papers Publ. micrographs Pers. commun. Previous paper Previous paper

Mollusca, Gastropoda, Pulmonata Arthropoda, Insecta, Orthoptera

This paper This paper

Anthophyta, Liliopsida, Liliales Anthophyta, Liliopsida, Cyperales

Publ. micrographs Publ. micrographs

Ref.

Cell type’

PS PS 1 2

3 4 5

PSISSIES PS/ES PS PS PS PS

PS

PS 6 7

M M

‘References: 1, Solari and Tres, 1967; Solari, 1969; Knibiehler et, al., 1981; Mirre and Knibiehler, 1985. 2, Barcellona and Brinkley, 1973. 3, E. Cnuve. 4, Wolf et al., 1987. 5, Wolf et al., 1988. 6, Gimenez-Martin and Stockert, 1970. 5, Gillies and Hyde, 1973. ‘PS, primary spermatocytes; SS, secondary sperrnatocytes; ES, early spermatids; M, microsporocytos.

present per nucleus, whereas several copies of the spherical body could occur in a single random section; (3) the nubecula infrequently associated with nucleoli, whereas the spherical body was always present within a nucleolus (Fig. 6). Another structure, the “fibrillar mass” in primary spermatocytes and spermatids of the wood lemming (Wolf et al., 19871, resembled the nubecula in its composition of “fine fibrillar material of low

density.” The structures otherwise differed in their relationship to the sex chromatin (the fibrillar mass was intimately associated with the XY pair, whereas the nubecula was not) and in the duration of their existence (the fibrillar mass persisted into spermiogenesis whereas the nubecula was present only during early pachytene). The fibrillar mass and the nubecula, therefore, probably are not related.

22

M.C. SC:HULTZ

Fig. 15. Low magnification micrograph of a rat stage I early pachytene spermatocyte in which the nubecula (arrowheads) is found against the nuclear envelope. x 13,100.

Fig. 17. Nubecula (arrowheads) in a stage I early pachytene spermatocyte, here adjacent to a round body that is surrounded by the rRNP particles of the granular component. x 35,400.

Fig. 16. Detail of the nubecula (N) in Fig. 15, showing its typical close relationship to the inner membrane of the nuclear envelope, as opposed to the ill-defined association with surrounding somatic chromatin. x 34,900.

Fig. 18. High magnification view of the nubecula (N) in a stage I early pachytene spermatocyte. It is composed of‘ approximately 12nm-wide fibrils (between arrowheads) that stain poorly by comparisun with the 18-nm-wide filaments of chromatin (arrows) a t its border. X

Some structures in somatic cells bore superficial resemblance to the nubecula. For example, weekly staining threads or filaments were described in the so-called “simple” nuclear bodies and in the fibrillar capsule of “complex” nuclear bodies in many somatic cells (Bouteille et al., 1974; Puvion and Moyne, 1981; Fitzgerald and Padykula, 19833. Unlike the nubecula, however, simple nuclear bodies were not connected to the nuclear envelope (Le Goascogne and Baulieu, 1977; Dupuy-Coin et al., 1982);and the nuclear envelope and

Y5,800.

fibrillar capsule of complex nuclear bodies were rarely seen in association (Weber et al., 1964; Yasuzumi et al., 1981). Other filamentous inclusions reported in somatic nuclei under normal (Seite et al., 19793 and pathological (Bingen and Kirn, 1975) conditions were distinct from the nubecula in their greater electron opacity and composition of 7 to 8-nm-wide filaments aligned in parallel arrays. An important feature of the nubecula was its transitory presence. It was observed only during stages I-

KX‘I’IIANLIC‘LEOLAI1STRUCTUItES I N SPERMATOCYTES

IV of the cycle of the seminiferous epithelium, i.e., for 3.2 days or approximately one-sixth of the meiotic prophase I in Sherman rats (Clermont et al., 1959). This was approximately the time when the coiled body disappeared from the nucleus-a time interpreted above as a period of active hnRNA synthesis. ACKNOWLEDGMENTS

This work was done with the support of a grant from the Medical Research Council of Canada to Dr. C.P. Leblond, while the author received the David Stewart Memorial Fellowship from McGill University and a Foundation Travelling Scholarship from the University of Queensland, Australia. LITERATURE CITED Harcellona, W.J., and B.R. Brinkley 1973 Effects of actinomycin D on spermatogenesis in the Chinese hamster. Biol. Reprod., NrY36349. Bendayan, M. 1981a IJltrastructural localization of nucleic acids by the use of enzyme-gold complexes. J. Histochem. Cytochcm., 29: 531-541. Hendayan, M. 1981b Electron microscopical localization of nucleic acids by means of nuclease-gold complexes. Histochem. J., 19: 699-710. Bernhard, W. 1971 Drug-induced changes in the interphase nuclcus. In: Advances in Cytnpharmacology. Vol. 1. F. Clementi and B. Ceccarelli, cds. Raven Press, New York, pp. 49-67. Bingen, A,, and A. Kirn 1975 Fibrillar bodies in hepatocyte nuclei during the course of toxic hepatitis produced by Frog Virus 3 in mice. J. Ultrastruct. Res., 50:167-173. Routeille, M., M. Laval, and A.M. Dupuy-Coin 1974 1,ocalization of nuclear functions as revealed by ultrastructural autoradiography and cytochemistry. In: The Cell Nucleus. Vol. 1. H. Busch, ed. Academic Press, New York, pp. 3-71. Clerrnont, Y., C.P. Leblond, and €3. Messier 1959 Duree du cycle de l’kpithelium seminal du rat. Arch. Anat. Microsc. Morphol. Exp.,

48:37-56. 1)upuy-Coin, A.M., M. Eouteille, P. Moens, and J.-G. Fournier 1982 ‘i’hree-dimensional distribution of nuclear organelles in measles virus induced pulykaryons. Biol. Cell, 43t55-68. Fakan, S., G. Leser, and T.E. Martin 1984 Ultrastructural distrihulion of nuclear ri bonucleoproteins as visualized by immunocytochemistry on thin sections. J. Cell Biol., 98t358-363. Fitzgerald, M., and H . A . Padykula 1983 Differing functional responses of simple and complex nuclear bodies in uterine luminal cndothelial cells following estrogenic stimuli. Anat. Rec., 205: 131-141. Friedlander, M., J. Gershon, and A. Rcinhartx 1976 Atypical cycle of the nucleolus in spermatocytes of the insect Locus~ornigraturicr. Cytobiolugie, 13r171-181. Gillies, C.B., and B.B. Hyde 1973 Intranucleolar bodies in maize pachytene microsporocytes. Hereditas, 74:137-140. Gimenez-Martin, G., and J.C. Stockert 1970 Nucleolar structure during the meiotic prophase in Alliuin cepa anthers. Z.%elltorsch. Mikrosk. Anat., 107r551-563. Goessens, G. 1984 Nucleolar structure. Int. Rev. Cytol., 87;107-168. Hermo, L., M. Lalli, and Y. Clermont 1977 Arrangcment of connective tissue components in the walls of the seminiferous tubules of man and monkey. Am. J. Anat., 148:433-446. HervBs, J.P., J. Villegas, D. Crespo, and M. I’afarga 1980 Coiled bodies in supraoptic nuclei of the rat hypothalamus during the postnatal pcriod. Am. J. Anat., 159r447-454. Kindci-man, N.B., and A. LaVelle 1976 A nucletJlus-associatedcoiled body. J. Neurocytol., 5545-550. Knibiehler, B., C. Mirre, M. Hartung, P. Jean, and A. Stahl 1981 Sex vesicle-associated nucleolar organizers in mouse spermatocytes: Localization, structure, and function. Cytngcnet. Cell. Genet., 31: 47-57. Lafarga, M., D. Crespo, and J. Villegas 1983a Nuclear inclusions in immature glial cells or the rat hypothalamus during the postnatal period. Anat. Ernbryol., 167.263-271. Lafarga, M., J.P. Hervds, M.C. Santa-Cruz, J . Villegas, and D. Crespo 19X3b the “act:essory body” o f Cajal in the neuronal nucleus. A light and electron microscopic approach. Anal. Embryol., 166: 19-30. Leblond, C.P., and Y. Clermont 1962 Definition ofthe stages of the

23

cycle of the seminiferous epithelium in tlic rat^. Ann. N.Y. Acad. Sci., 55r548-573. Le Goascogne, C., and E.-E. Baulieu 1977 Hormonally controlled “nuclear bodies” during development of the prepuberal rat uterus. Biol. Cell, 3Or195-206. Matsushima, S., Y. Sakai, I. Aida, and R.J. Reiter 1984 Nuclear and cytoplasmic inclusion bodies in pinealocytes of the cotton rat, Sigmodon hispidust an electron microscopic study. J. Pineal Res., 1:293-304. Mirre, C., and B. Knihiehler 1985 Ultrastructural and functional variations in the apermatid nucleolus during spermiogenesis in the mouse. Cell Iliff., 1 6 5 - 6 1 . Monneron, A., and W. Bernhard 1969 Fino structural organization of the interphase nuclcus in some mammalian cells. J. Ultrastruct. Res., 27.266-288. Moreno Diaz de la Espina, S., M.C. Risueno, and F.J. Medina 1982 Ultrastructural, cytochemical and autoradiographic characterization of coiled bodies in the plant cell nucleus. Biol. Cell, 44: 229-238.

Paniagua, R., M. Nistal, P. Amat, and M.C. Rodriguez 19% Ultrastructural observations on nucleoli and related structures during human spcrmatogenesis. Anat. Embryol., 174:301-306. Puvion, E., and G. Moyne 1981 In situ localization of RNA structures. In: The Cell Nucleus. Vol. VIII. Nuclear Particles, Part A, H. Busch, ed. Academic Press, New York, pp. 59-1 15. Keed, R., J. Griffth, and T. Maniatis 1988 Purification and visualization of native spliceosomes. Cell, 63t949-961. Sananes, N., and C. IJe Goascogne 1976 Decidualization in the prepubertal rat uterus. Differentiation, Sr133-144. Schultz, M.C. 1986 Characterization of the “round body” and other struclures associated with the nuclcolus in male germinal cel Is. Ph.D. Thesis, McGill University, Montreal, Quebec, Canada. Schultz, M.C. 1989 Ultrastructural study of t,he coiled body and a new inclusion, the “mykaryon,” in thc nucleus of the adult rat Sertoli cell. Anat. Rec., 225;21-25. Schultz, M.C., and C.P. Leblond 1990 I. Nucleolar strueturc and synthetic activity during moiotic prophase and spermiogcncsis in the rat. Am. J. Anat., this issue. Schultz, M.C., L. Hermo, and C.P. Leblond 1984 Structure, development, and cytochemical properties of the nucleolus-aRsnciated “round hody” in the rat spermatocytcs and early sperniatids. Am. J. Anat., I71:41-57. Seite, R., M.J. Pcbusque, and M. Vio-Cigna 1982 Argyrophilic proteins on coiled bodies in sympathetic neurons identified by AgNOR procedure. Biol. Cell, 46t97-100. Seite, R., J . Vuillet-Luciani, R. Zerbib, C. Cataldo, J . Escaig, M.J. Pehusque, and A. Autillo-Touati 1979 Three-dimensional organization of tubular and filamentous nuclear inclusions and associated struct.ures in Yympathetic neurons as revealed by serial sections and tilting experiments. J. Ultrastruct. Res., 69:21 1-231. Silverman, L., and D. Glick 1969 The reactivity and staining of tissue proteins with phosphotungst.ic acid. J . Cell Biol., 40;761-767. Solari, A.J. 1969 The evolution of the ultrastructure of the sex chromosomes (sex vesicle) during the meiotic prophase in mouse spermatocytes. J. IJltrastruct. Res., 272289-305. Solari, A.J., and L. Trcs 1967 The localization of nucleic acids and the argentaffin substance in the sex vesicle of mouse spermatocytes. Exp. Cell Res., 47386-96. Stockert, J.C., G. Gimenez-Martin, and J.M. Sogo 1970 Nucleolus and synagtonemal complcxes in pachytene meiocytes of Alliom ceppu. Cytobiolugie, 2.235-250. Weber, A,, S. Whipp, E. Uscnik, and S. Frommcs 1964 Structural changes in lhe nuclear body in the adrenal zona fasciulat,a of the calf following the administration of ACTH. J. Ultrastruct. Res., 11,564-576. Williams, M.A., .J.A. Kleinschmidt, G . Krohne, and W.W. Franke 1982 Argyrophilic nuclear and nucleolar proteins of Xrrzopus laeuis oocytes identified by gel electrophoresis. Exp. Cell Res., 137: 341-351. Wolf, K.W., K. Baumgart, and H. Winking 1988 Meiotic association and segregation of the achiasrnatic giant sex chromosomes in the male field vole fMicrotus ugr Chromosonia, 97t124 -133. Wolf‘, K.W., H. Winking, and K. a 1987 Relationship between nucledi and sex chromosomes during meiosis of the male wood lemming Myopus schisticolorr A fine structure study. Biol. Cell, 60:16-24. Yasuzumi, G., N . Yabumoto, T. Shirai, Y. Nakai, and F. Yasuzunii 1981 Functions of nuclear bodie revealed by ultrastructural autoradiography and cytochemistry. Experientia, 37t1072-1073.