THE AMERICAN JOURNAL OF ANATOMY 189:1-10 (1990)
Nucleolar Structure and Synthetic Activity During Meiotic Prophase and Spermiogenesis in the Rat MICHAEL C. SCHULTZ AND C. P. LEBLOND Department of Anatomy, McCill University, Montreal, Quebec, Canada H3A 2B2
ABSTRACT The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli was examined in rats sacrificed 1 hr after intratesticular injection of 'H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords-a nucleolonema-within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extrunucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 'H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the @ 1990 WILEY-LISS, INC.
second maturation division do not synthesize rRNA. INTRODUCTION
The radical transformations occurring in cytoplasm and nucleus during spermatogenesis have overshadowed the changes taking place in the nucleolus. Yet i t is known that nucleoli are modified in several ways during meiotic prophase and spermiogenesis. In the rat, for example, they gradually enlarge during the pachytene stage (Daoust and Clermont, 1955; Urena and Solari, 1970; Stefanini et al., 1974); while a t the end of this stage, their number decreases (Daoust and Clermont, 1955) and the dense fibrillar component (pars fibrosa) of the nucleolus segregates from the granular component (pars granulosa) (Stefanini et al., 1974; Leblond, 1981). In spermatids, the nucleoli are small, compact structures which eventually disappear altogether (Leblond, 1981). The role of nucleoli is to synthesize the main RNA components of ribosomes, i.e., the 5.8, 18, and 28s ribosomal RNAs (rRNA) (Jordan, 1978). The synthesis of these species may be examined by radioautography soon after 3H-uridine incorporation since the silver grains that appear over the nucleolus are then due to newly formed rRNA (Fakan and Puvion, 1980; Uddin et al., 1984). The first attempts at 3H-uridine radioautography of spermatocyte nucleoli in locusts (Das et al., 1965) and hamsters (Utakoji, 1966) revealed no reaction, suggesting that rRNA transcription was inhibited during male meiotic pro hase Later, however, .: Stefanini et al. (1974) injected ! H-uridine directly into the rat testis and examined rRNA synthesis by LM radioautography. They observed a reaction over spermatocyte nucleoli, with a peak at mid-pachytene, but a long exposure was required. Thus there would be a rather low rate of rRNA synthesis. Soderstrom (1976) examined rRNA synthesis in the rat and concluded that this activity was limited in mid-pachytene cells. Significant rRNA synthesis, however, was reported in mouse spermatocyte nucleoli, except a t late pachytene (Kierszenbaum and Tres, 1974). Finally, a careful study of cultured human spermatocytes (Tres, 1975) led to the conclusion that 3H-uridine incorporation by
Received October 24, 1988. Accepted March 26, 1990. Address reprint requests to Dr. C . P. Lehlond, Department of Anatomy, McGill University, Montreal, Quehec, Canada. Dr. M.C. Schultz's present address is Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
2
M.C. SCHTJLTZ A N I ) C.P. LELILOND
nucleoli was low a t leptotene, reached a peak a t late zygotene, and declined a t late pachytene. In the hope of unifying the scattered information on the behavior of nucleoli during male meiosis and spermiogenesis, their ultrastructure and ’H-uridine radioautography were examined in the rat under in vivo conditions. The meiotic prophase was divided into three phases: 1)preleptotene, leptotene, and zygotene; 2) early and mid-pachytene; and 3) late pachytene, diplotene, and diakinesis. Nucleoli were also examined in secondary spermatocytes and spermatids a t early steps of development. For the ultrastructural study, “random” sections of the testis, that is, sections collected without regard to serial order and orientation, were used initially. Later, “serial” sections of nuclei were prepared to measure the size of nucleoli. These sections also revealed that primary spermatocytes may contain not only nucleoli which have the usual complement of fibrillar centers, fibrillar component, granular component, and interstitial spaces, but also structures composed of a granular component independent of any nucleolus; these structures will be referred to as extranucleolar granular elements. In parallel with the ultrastructural study, the incorporation of 3H-uridine into the nucleolus was assessed in vivo by EM radioautography of random sections. The abundance of silver grains over nucleoli, used as a n index of the rate of rRNA synthesis was related t o the changing features of nucleoli in spermatocytes and spermatids.
outline of each cross section was traced onto transparency sheets, and the tracings were collectively weighed on a Sartorius analytical balance. Since the weight was proportional to the actual volume, this volume was calculated from the final magnification of the micrographs ( x 24,000) and the mean section thickness (60 nm, estimated by direct measurement of re-embedded sections cut a t 90 degrees to the original plane of section). In the case of the largest nucleolus in a given nucleus, not only the total volume, but also that of its granular component was measured by the cut-out weighing method. The volume of the rest of the nucleolus, that is fibrillar centers and fibrillar component, was obtained by subtracting the volume of the granular component from the total volume. Next, the proportion of the granular component occupied by interstitial spaces was determined by the point-hit method (Elias and Hyde, 1983) using a grid lattice containing 56 points, with the dist,ance between points representing three times the average diameter of the ribonucleoprotein granules (RNP) of the granular component. Finally, the volume of each extranucleolar granular element was measured by the cut-out weighing method in serially cut nuclei. ’H-Uridine Radioautography
To examine the incorporation of “H(5’1-uridine into RNA by the nucleoli of spermatocytes and spermatids, adult rats were anesthetized and the ri h t testis was exposed for direct injection of 0.25 mCi of, H(5’buridine MATERIALS AND METHODS (specific activity, 26.0 Ci/mM; New England Nuclear) in Preparation and Analysis of Serial Sections physiological saline. The skin was then sutured, and 1 Testes were obtained from adult Sherman rats h r later the testes were fixed by retrograde perfusion as weighing 190-380 gm. The fixative, 5% glutaralde- above using 2.5% glutaraldehyde. Tissue blocks were hyde in 0.1 M sodium cacodylate buffer at pH 7.3, was then immersed in primary fixative for 2 hr, rinsed, and given by retrograde perfusion into the abdominal aorta postfixed for 2 h r in 1%osmium tetroxide. Processing for after clamping the aorta above the kidney and cutting EM radioautography was done as previously described the renal veins (Vitate et al., 1973). The tissue blocks (Schultz e t al., 19841, using either a 2-month or, in most were then immersed for 1 hour into reduced osmium, cases, a 6-month exposure. Preliminary light-microthat is a 1:l mixture of 2% aqueous osmium tetroxide scopic radioautographs showed widely different radioand 3% potassium ferrocyanide (Karnovsky, 1971). activities in the various parts of the testis. Hence, the The samples were embedded in Epon, and the sections relative labeling intensity over the various cell types were routinely stained with uranyl and lead salts. was assessed subjectively in comparable areas (see TaSerial sections of nuclei were examined in primary ble 3 below). In one case, labeling intensity was quanspermatocytes a t specific stages of the cycle of the se- titated in four adjacent tubules surrounding a large miniferous epithelium, a s classified by the method of blood vessel which presumably provided them with a Leblond and Clermont (1952). The serial sections were similar amount of radioactive precursor. Silver grains mounted on Formvar-coated slotted grids according to were counted over the fibrillar component (with fibrilStevens et al. (1980). Thus one set included 93 consec- lar centers included), whose area was determined by the utive sections showing part of three tubules a t stages I, cut-out weighing method as above. The grain counts VII, and XII, respectively. The profile of each nucleus were expressed per unit area and averaged. under study was photographed in every serial section using a Philips 300 or a 400T electron microscope. In other series mounted on wide-mesh grids, larger sets of sections were obtained, in which a t least every fifth section through a particular nucleus was photographed. In each serially sectioned nucleus, the volume of the largest nucleolus and the largest extranucleolar granAhhrruiations hetorochromatin ular element (identified by surveying the sections) was Chr fibrillar component determined as follows. The total area of a structure in rfc fibrillar center each cross section was determined by the “cut-out g granular component weighing” method (Elias and Hyde, 19831, that is, the NE nuclear envelope
NTJCLEOLUS I N RAT SPP:ItMATOC;ENl(’ CELIA
Fig. 1. Nucleolus in aplaleptolerrr primary spermatocyte (stage VII). The cytoplasm at lower left i s separated by the nuclear envelope from the nucleus, which includes some heterochromatin and, in its center, a nucleolus. Within lhe nucleolus, a light-gray fibrillar center is associated with cords of the fibrillar component. Extending above it is the granular component; i t is formed of patches from which extend short, broad cords; patches and cords arc composed of ribonucleoprotein particles. X 48,500.
3
Fig. 2. Nucleolus in a n eurLy pachytene spermatocyte at stage I. A poorly limited fibrillar center is continuous with cords of the Gbrillar component. The granular component is dispersed in patches (upper right) and slender cords (lower) that form a reticulated network. The interstitial spaces separating the cords of granular component are more lightly stained than the nucleoplasm proper. x 48,500.
Fig. 3.
5
NUCLEOLUS I N RAT SPERMATO(;ENIC CELLS
RESULTS Spermatocyte and Spermatid Nucleoli, As Seen in Random Sections First phase of meiosis
In preleptotene spermatocytes (stage VII of the cycle of the seminiferous epithelium), nucleoli are relatively small (Fig. 1).They include fibrillar centers which appear as pale gray structures, a fibrillar component consisting of few dense cords, and a granular component made up of few patches and cords separated by narrow interstitial spaces. At leptotene and zygotene (stages VIII-XIII), spermatocyte nucleoli show little change in size and appearance. Second phase of meiosis
In early pachytene spermatocytes, i.e., from stage XIV of one cycle to stage IV of the next cycle, there is gradual enlargement of the nucleolus. Fibrillar centers remain small, but the cords of fibrillar component appear more numerous and more loosely organized than in the first phase of meiosis. The granular component enlarges; its patches and cords become numerous, but narrow; they spread out into a tridimensional network, while the interstitial spaces between them widen and appear lighter than the rest of the nucleoplasm (Fig. 2). During mid-pachytene, which extends from stage V to VIII of a cycle, nucleoli occupy a large fraction of the nuclear volume. Fibrillar centers are small, pale-gray structures located at a distance from one another; they remain connected through cords of the fibrillar component. The loosening of fibrillar and granular components noted at early pachytene is enhanced, so that both spread out over a large domain (Fig. 3). In particular, the granular component forms an extensive network composed of numerous small patches interconnected by narrow cords between which are large, light interstitial spaces. Third phase of meiosis
In late pachytene, which extends from stage IX t o XI1 of the cycle, there is a progressive condensation and segregation of nucleolar components. Fibrillar centers change t o compact, electron-dense masses located within an electron-lucent halo (Fig. 4). They are surrounded by dense, thick cords of the fibrillar component which are increasingly separated from the granular component. The patches and cords of the latter agglomerate, leaving only small interstitial spaces between them (Fig. 4). At diplotene (stage XIII) and diahinesis (stage XIV), the condensation of the fibrillar component is accentuated (Fig. 5), and its segregation from the granular component is completed.
Secondary spermatocytes and spermatids
Nucleoli have not been seen during the first maturation division, but reappear in secondary spermatocytes. They are then composed of a fairly solid fibrillar component containing a diffuse fibrillar center, while the granular component condenses on one side of it (not shown). During the second maturation division, nudeoli are again not seen. However, the step 1 spermatids that arise from the final division of spermatogenesis have a small, ovoid nucleolus. It is composed of a distinct, solid fibrillar component containing a diffuse fibrillar center and, as in secondary spermatocytes, the small granular component is segregated from the fibrillar component (Fig. 6 ) .The granular component is lost by the end of step 6 . At step 7, small cavities appear in the fibrillar component and make it difficult to ascertain whether a fibrillar center still exists. At step 8, the nucleolus is exclusively composed of fibrillar component. By step 9, no trace of the nucleolus can be identified. Features of Nucleoli Observed in Serial Sections
Serial sections reveal that, in addition to normal nucleoli in which fibrillar centers and fibrillar and granular components are associated, there may be extranucleolar granular elements (Table 1).The data indicate that, as the cell progresses from leptotene to pachytene, the number of nucleoli decreases, while that of extranucleolar granular elements increases. At late pachytene, however, no extranucleolar granular elements have been observed, except for a single small one. For further analysis, one nucleus has been selected at each stage, and data are recorded for its largest nucleolus and its largest extranucleolar granular element (Table 2). The volume of complete nucleoli increases proqressively from 0.22 km3 at preleptotene to 3.43 km' at late pachytene. Component structures, partic-
TABLE 1. Number of nucleoli and extranucleolar granular elements as observed in serial sections during meiosis' ~
Phase Preleptotene Early pachytene Mid pachytene
~
Approximate No. ExtraNo. of Percent nu c1ear nuclei of nucleus granular examined cut seriallv Nucleoli elements 1 57 7 0 2 64 3 7 4
78 79 79 93
92 Late pachytene ~
Fig. 3. Nucleolus of a mid-pachytene primary spermatocyte (stage V11). A fibrillar center appears homogeneous and well limited. The fibrillar component i s distributed in cords that are associated with the center or intermingle with cords of the granular cumponent. The latter forms a large, reticulated network extending uver most of the micrograph. The patches and cords of the netwurk are separated by pale interstitial spaces. x 34,900.
3
42
59 73
2 2 1 3 2 2
2 3
7 1 6
5 4
0 0 1
'Only the preleptotene nucleus was completely included in the series. It IS estimated that early, mid- and late pachytene nucleoli occupy, respectively, 90%, 75-907'0, and 50-75%) of each nucleus. Hence, except for the prcleptotene nucleus, the numbers in the last two columns are underestimates.
M.C. SCHIILTZ AND C.P. LEHLOND
Fig. 4. Nucleolus of a late pachyime sperrnatocyte (stage XII) containing a condensed fibrillar center outlined by a light space and surrounded by dense cords of the fibrillar component that tend to separate from the granular component. The latter is composed of closely packed patches and cords, with only small interstitial spaces. x 52,500.
lar component (fg) in which some granules are evident. The section by-passed the granular component, of which only the edge is cut tangentially. ~ 5 2 , 5 0 0 .
Fig. 5. Nucleolus in a primary spermatocyte a t diakinesis. A fibrillar center is surrounded by dense, sharply outlined cords ofthe fibril-
Fig. 6. Nucleolus in a step 1 spermatid. This nucleolus is small. It consists of a compact fibrillar component segregated from the granular component. The fibrillar component includes a slightly paler area which is presumed to be a diffuse fibrillar center (arrowheads). x 52,500.
ularly the granular, also increase; the interstitial spaces enlarge up to mid-pachytene and decrease at late pachytene (Table 2). The extranucleolar granular elements at early and mid-pachytene consist of ribonucleoprotein (RNP) granules arranged in loose patches and cords similar to those in the granular component of nucleoli at the same stage, but their volume is highly variable. Thus,
the large extranucleolar granular element (0.80 m3) recorded at early pachytene is larger than the biggest nucleolus in the same nucleus (0.48 +m3) (Table 2). Another one incorporates a round body, as often does the granular component of nucleoli (Schultz et al., 1984). Extranucleolar granular elements may be minute; thus, a small one has been found which is embedded within the condensed XY pair. Finally, at late
NUCLEOI~USIN RAT SPEKMATOGENIC CELIJS
7
TABLE 2. Size of nucleoli and extranucleolar granular elements measured in serial sections in one nucleolus at each phase of the rat meiotic prophase and in a young spermatid'
Prel eptotene Early pachytene Mid pachytene Late pachytene Step 1 spermatid
Stage VII I VII XI1 I
Number of nucleoli Per nucleus 7 3 3
Whole nucleolus 0.22 0.48
1.91
2
3.43
1
0.09
Volumes in largest nucleolus (pm3) Center Granu+ fibrillar lar comcompoponent nent 0.04 0.15 0.15 0.14 0.26 0.64 0.47 2.57 0.06 0.03
Interstitial spaces 0.03 0.19 1.01
0.38 -
Extranucleolar granular elements No. Volume of Per largest one nucleus (pm3 0 7 0.80 5 1.20 0 -
-
'The complete and incomplete nucleoli were from the same nucleus a t each stage, except at mid-pachytene, where they came from two different nuclei.
TABLE 3. Label incorporation by nucleoli and nucleoplasm during meiotic rophase and spermiogenesis, (1 hr) after ..p H-uridine injection into rat testis
Leptotene
Early pachytene Mid-pachytene Diakinesia Earlv mermatid
Staee of cvcle IX XIV,II V1,VII XI11 I
Intensity of labeling' Nucleolus Nucleoolasm +I+ + +
++ ++++ + I0
++ ++-I ++ ++
Over the nucleoplasm, silver grains are present at all stages of meiosis (Figs. 7, 8) as well as in young spermatids (Fig. 9). The reaction increases to a peak at mid pachytene and decreases thereafter, but remains definite in young spermatids (Table 3). DISCUSSION Structural Changes in the Nucleolus of Spermatocytes and Spermatids
The early primary spermatocytes from preloptotene to zygotene had small nucleoli. The limited but precise 'Relative intensity is given in ascending order from 0 (when labeling data in Table 2 indicated that, in the course of early is absent or doubtful) to a maximum of + + i t (when it is intense). and mid pachytene, nucleoli formed an extensive, lacy The reaction over the nucleolus is attributed to newly-formed ribosomal RNA. The reaction over the nucleoplasm is attributed to newly- nucleolonema and thus gradually enlarged to about 9 times their initial volume, due in part to an increase in formed messenger and transfer RNA. the RNP granules (about fourfold) and interstitial spaces (over 30 fold) of the granular component. In the pachytene, the single small extranucleolar granular el- meantime, extranucleolar granular elements of variement (0.05 pm3) is composed, as in nucleoli at that ous sizes made their appearance. Such structures had stage, of a closely packed aggregate of RNP particles. been previously observed in human spermatocytes at leptotene (Stahl et al., 1983; Paniagua et al., 1986), that is at an earlier phase than in the rat. The extraEM Radioautographic Survey of nucleolar granular elements were likely to be frag3H(5')-Uridine Incorporation ments of that granular component that had been In testicular tissue fixed 1 hr after injection of 3H- cleaved from complete nucleoli, presumably as a result uridine, a radioautographic reaction is observed over of the chromosomal movements observed by Parvinen the nucleoli of primary spermatocytes (Table 3). The and Soderstrom (1976) in rat spermatocytes from late reaction is moderate during the first phase of meiosis, leptotene to mid-pachytene. but increases gradually during the second phase, being By late pachytene, the three main nucleolar parts fairly strong at early pachytene and intense through- condensed gradually. Fibrillar centers became particout mid-pachytene up to the end of stage IX (Fig. 7). At ularly dense. The fibrillar and granular elements segeach phase, the reaction is essentially localized over regated, while the interstitial spaces present within the fibrillar component. During late prophase and fol- them decreased markedly. Meanwhile, the extranuclelowing stages (third phase of meiosis), nucleolar reac- olar granular elements disappeared more or less comtivity is markedly reduced. Thus, at diakinesis, there pletely. It was likely that they had rejoined the nucleare rare silver grains over the aggregated cords of the oli, and thus contributed to their enlargement at late fibrillar component and none over other nucleolar pachytene. parts (Fig. 8 ) . In early spermatids, nucleoli are not laNucleoli were small and compact in secondary sperbeled (Fig. 9, Table 3). Some quantitative measure- matocytes and even more so in spermatids1 (Schultz et ments confirmed these conclusions. For instance, in al., 1984);they disappeared by step 8 of spermiogenesis. four adjacent tubules that appeared evenly labeled, counts of the mean number of silver grains per pm2 of fibrillar component were as follows: 44.6 in early pachytene (stages I and 111, 127.3 in mid pachytene from published photographs, compact, segregated nucleoli (stage VII), 4.5 at diakinesis (stage XIV), and a back- are'Judging present in the young spermatids of hamster (Barcellona and Brinkground count in secondary spermatocytes (stage XIV) ley, 1973), mouse (Krimer and Esponda, 1979; Czacker, 1984, 19x5; as well as in step 1 and 2 spermatids (stages I and 11). Mirre and Knibiehler, 1985), and guinea pig (Ohtomo, 1981). 0
Figs. 7-9.
Changes in rRNA Synthesis in Spefmatocytes and Spermatids
The role of the ribosomal genes present in fibrillar centers (reviewed in Perry, 1981; Hugle et al., 1985; Aris and Blobel, 1988) is to produce a primary transcript, the 45s rRNA, which passes into the fibrillar component, while associating with protein. It is then split into 1)a n 18s rRNA which leaves the nucleolus to be built into the minor (405) ribosome subunits, and 2) a 32s rRNA, soon split into 28s and 5.8s rRNA, both of which are presumed to be part of the granular component; they will eventually be built into the major (60s) ribosome subunits. When, a s in the present investigation, radioautographic reactions are observed over the fibrillar component soon after “H-uridine injection, they are attributed to newly formed 45s rRNA and its immediate progeny; and therefore, can be used a s a n index of rRNA synthesis (Fakan and Puvion, 1978; Uddin et al., 1984). The intensity of radioautographic reaction over spermatocyte nucleoli indicated that rRNA synthesis was low from preleptotene to zygotene, gradually increased during early pachytene to a maximum a t midpachytene, and then declined during late pachytene to become negligible a t diplotene. No significant rRNA synthesis occurred in the nucleoli of secondary spermatocytes and spermatids. Incidentally, the intensity of silver staining by nucleoli which, in the past, had been taken as a n index of rRNA synthesis was more likely to indicate the presence of particular proteins (Goessens, 1984). Thus, even though spermatid nucleoli were stained with silver (Hofgartner et al., 1979), they showed no evidence of rRNA synthesis. Interprefation of the Results
When nucleoli were examined in various types of differentiating cells (blood cells-Busch and Smetana, 1970; keratinocytes and sebaceous cells-Karasek et al., 1972, 1973; epithelial cells in alimentary tractAltmann and Leblond, 1982; Lee, 1985), it was con-
~~
~~
Fig. 7. Radioautugraph of the nucleolus from a spermatocyte ob-
served at about the end of mid-pachytene (stage 1x1 fixed I hr after intratesticular injection of “H-uridine. Fibrillar and granular components are prominent, hut there is some decrease in size of the interstitial spaces. Radioautographic silver grains are numerous over the fibrillar component; while over the granular component grains are rare, except close to the fibrillar component. There is a fair reaction throughout the nucleoplasm. XY, Condensed sex pair. x 30,000. Fig. 8. Radioautograph of the nucleolus from a primary spermatocyte at diakrnesis (stage XIVi 1 hr after %IH-uridineinjection. The fibrillar component which includes compact fibrillar cunters (arrows) is segregated from the condensed granular component which surrounds a “round body” (KR) with a large vacuole in thc center. Silver grains are rare over the fibrillar component and may be due to background fog. No silver grains arc present over the granular component. The reaction over the nucleoplasm is moderate. x 20,400. Fig. 9. Radioautograph of the nucleoluv from a step 1 spermatid 1 hr after 3H-uridine injection. The fibrillar component encloses a faintly visible fibrillar center (arrowheads) and a large round body (RBI. The scanty granular component includes only a few RNP particles. No silver grains are seen on any part of the nucleolus. However, thcre is a moderate reaction in the nucleoplasm. x 48,000.
cluded that undifferentiated proliferative cells had active nucleoli, characterized by a large, highly reticulated network, the nucleolonema, whereas fully differentiated cells usually had less active or inactive nucleoli, which were often small and compact. For example in r a t small intestine, a s columnar cells differentiated during their migration from crypt base to villus top, nucleolar volume decreased by a factor of 16, while fibrillar and granular components gradually condensed and became segregated (Altmann and Leblond, 1982). In the meantime, counts of silver grains over nucleoli in 3H-uridine radioautographs were high in crypt base, gradually decreased during the migration from crypt to villus, and were a t background level in villus top (Uddin et al., 1984). Thus, in general, the rRNA synthetic rate decreased in parallel with nucleolar size. During early and mid-pachytene, nucleolar volume increased and a nucleolonema-like network developed, while rRNA synthesis increased markedly. These findings were indicative of a n activation of ribosomal gene transcription, in contrast to the previous view that rRNA synthesis was lacking (Das et al., 1965; Utakoji, 1966) or low (Stefanini et al., 1974) during male meiosis. The ribosomal gene activation during early and mid-pachytene was associated with a fourfold increase in RNP granules (Table 2). In the case of somatic mitosis, only a doubling of these granules was observed during the cycle (Lepoint and Goessens, 1982). The next phase of spermatocyte development, extending from late pachytene to the first maturation division, was characterized by a decrease in radioautographic reaction, which fell to a negligible level during diplotene. Meanwhile, there was condensation and segregation of nucleolar components, but no decrease in nucleolar size, or rather a n increase, attributed in part to aggregation of extranucleolar granular elements as mentioned earlier and in part to the possible fusion of whole nucleoli (as suggested by the figures in Daoust and CLermont, 1955). Nevertheless, the waning of rRNA synthesis and the segregation of nucleolar components demonstrated the inactivation of ribosomal gene transcription. The shutdown was similar to t h a t observed at metaphase and anaphase in the mitotic cycle of somatic cells (Fan and Penman, 1971; Lepoint and Goessens, 1978). Nucleolar rRNA synthesis was promptly restored at the end of somatic mitoses, but this was not the case in the secondary spermatocytes arising from the first maturation division nor in the spermatids arising from the second. The level of rRNA synthesis in spermatids had been previously examined by biochemical studies of spermatids separated by centrifugation. In contrast to our observations, Geremia et al. (1978) reported that “quasi-homogeneous” preparations of round spermatids from mouse tubules labeled with ,’H-uridine in vitro synthesized as much rRNA as mid-late pachytene spermatocytes. However, Meistrich e t al. (1981) isolated 985% pure pachytene spermatocytes and 93% pure round spermatids after intratesticular injection of H-uridine to rats and found that pachytene cells were active in rRNA synthesis, but round spermatids were not, in agreement with our findings. Finally, Kierszenbaum and Tres (1974, their Fig. 9) depicted in spermatid nuclei a dense spherical component which showed
10
M.C. SCHIJLTZ AN11 r.1’.LEKLONI)
no reaction in 3H-uridine radioautographs; our identification of this component as a nucleolus was in accord with the conclusion that spermatid nucleoli did not synthesize rRNA. In conclusion, the activation of ribosomal genes in nucleoli during early and mid pachytene is followed by inactivation just before the two meiotic divisions take place. Unlike what happens after somatic mitosis, there is no significant reactivation of ribosomal genes in the young spermatids arising from meiosis, ACKNOWLEDGMENTS
The first author successively received a David Stewart Memorial Fellowship from McGill University and a Foundation “ravelling Scholarship from the University of Queensland, Australia. Funding for the project was provided through a grant from the Medical Research Council of Canada. Critical reading of the manuscript by Dr. Y. Clermont is gratefully acknowledged. Ms. Sonia Bujold is thanked for her excellent technical assistance throughout the course of the work reported in this and the companion article. LITERATURE CITED Altmann, G.G., and C.P. Leblond 1982 Changes in the size and structure of the nucleolus of columnar cells during their migration from crypt base to villus top in ratjejunum. J. Cell Sci., 66.83-99, Aris, d.P., and G. Blobel 1988 ldentification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol., 107~17-31. Barcellona, W.J., and B.R. Brinkley 1973 Effects of actinomycin D on spermatogenesis if the Chinese hamster. Biol. Reprod., 13:335349. Busch, H., and K. Smetana 1970 The Nucleus. Academic Press, New York. Czaker, R. 1984 Observations on the dynamics of argyrophilic nucleolar material in the nuclei of mice spermatids. .Experientia, 40: 960-963. Czaker, R. 1985 Ultrastructural observations on nucleolar changes during mouse spermiogenesis. Andrologia, 17:42-53. Daoust, R., and Y. Clermont 1955 Distribution of nucleic acids in germ cells during the cycle of the seminiferous cpithelium. Am. J. Anat., 96:255-283. Das, N.K., E.P. Siegel, and M. Alfert 1965 Synthetic activities during spermatogenesis in the locust. J. Cell Hiol., 25:387-395. Elias, H., and U.M. Hyde 1983 A Guide to Practical Stereology. S. Karger, Basel. Fakan, S., and E. Puvion 1980 The ultrastructural visualization of nuclcolar and extranucleolar RNA synthesis and distribution. Int. Rev. Cytol., 65:255-299. Fan, H., and S. Penman 1971 Regulation of synthesis and processing of nucleolar components in metaphase-arrested cells. J. Mol. Biol., 5Y:27-42. Geremia, R., A. D’Agostinm, and V. Monesi 1978 Biochemical evidence of haploid gene activity in spermatogenesis of the mouse. Exp. Cell tles., lIlt23-:30. Goessens, G. 1984 Nucleolar structure. Int. Rev. Cytol., R7;107-158. Hofgartner, F.J., M. Schmid, W. Krone, M . T ~Zenses, and W. Engel 1979 Pattern of activity of nucleolus organizers during spermatogenesis in mammals as analysed by silver-staining. Chi-omosoma, 71~197-216. Hiigle, B., R. Hazan, U. Scheer, and W.W. Franke 1985 Localization of ribosomal protein S1 in the granular component ofthe interphase nucleolus and its distribution during mitosis. J. Cell Biol., 100: 873-886. Jordan, E.G. 1978 The Nucleolus. Carolina Biology Readers, Burlington, North Carolina. Karasak, J., K. Smetana, A. Hrdlicka, T. Dubinin, 0. Hornak, and W. Ochlert 1972 Nuclear and nucleolus ultrastructure during the
late stages of normal human keratinocyte maturation. Br. J. Dermatol., 86331-607. Karasak, J.,A. Hrdlicka, and K. Smetana 1973 Studies of the nuclear and nucleolar ultrastructure in differentiating and differentiated human sebaceous cells. J. Ultrastruct. Res. 42234-243. Karnovsky, M.J. 1971 Use of ferrocyanide reduced osmium tetroxide in electron microscopy. J. Cell Biol. (Proc.),5Zt14fja. Kierszenbaum, A.L., and L.L. Tres 1974 Nucleolar and perichromosoma1 RNA synthesis during meiotic prophase in the mouse testis. J. Cell Biol., 60~39-53. Kierszenbaum, A.L., and L.L. Tres 1975 Structural and transcriptional fcatures of the mouse spermatid genome. J. Cell Biol., 65: 258-270. Krimer, U.B., and P. Esponda 1979 Nucleolar fibrillar centers in mouse spermatid nucleoli. Eur. J. Cell Biol., 20:156-158. Leblond, C.P. 1981 The life history of cells in renewing systems. Am. J. Anat., 160;113-158. Leblond, C,.P., and Y. Clermont 1952 Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N.Y. Acad. Sci., 55~548-573. Lee, E.R. 1985 Dynamic histology of the antral epithelium in the mouse stomach. Ill. Ultrastructure and renewal of pit cells. Am. J. Anat., 172225-240, Lepoint, A., and G. Goessens 1978 Nucleogenesis in Ehrlich tumor cells. Exp. Cell Res., 117:89-94. Lepoint, A,, and G. Goessens 1982 Quantitative analysis of Ehrlich tumor cell nucleoli during interphase. ESP. Cell Res., 137:456459. Meistrich, M.L., d. Longtin, W.A. Brock, S.R. Grimes, Jr., and M.L. Mace 1981 Purification of rat spermatogenic cells and preliniinary biochemical analysis of these cells. Biol. Reprod., 25.10651077. Mirre, C., and H. Knihiehler 1985 Ultrastructural and functional variations in the spermatid nucleolus during spermiogenesis in the mouse. Cell Diff., 16~51-61. Ohtomo, K. 1981 Ultrastructural changes in the spermatid nucleolus during spermiogcnesis in the guinea pig. Gamete Res., 4:387394. Paniagua, R., M. Nistal, P. Amat, and M.C. Rodriguez 1986 Ultrastructural observations on nucleoli and related structures during human spermatogenesis. Anat. Ernbryol., 174:301-306. Parvinen, M., and K.-0. Soderstrom 1976: Chromosome rotation and Cormation of synapsis. Nature (Lond.),260.534-535. Perry, R.P. 1981 RNA processing comes of age. *J. Cell Biol., Y1:28a38s. Schultz, M.C., L. Hermo, and C.P. Leblond 1984 Structure, development, and cytocheniical properties of the nucleolus-associated “round body” in rat spermatocytes and early spermatids. Am. J. Anat., 171:41-57. Soderstrom, K . 4 . 1976 Characterization of RNA synthesis in midpachytene spermatocytes of the rat. Exp. Cell Res., 102r237-24. Stahl, A,, J.M. Luciani, M. Hartung, M. Devictor, J.L. BergeLafrance, and M. Guichaoua 1983 Structural basis for Robertsonian translocations in man: association of ribosomal genes in the nucleolar fibrillar center in meiotic spermatocytes and oocytes. Proc. Natl. Acad. Sei. USA, 805946-5950. Stefanini, M., C. de Martino, A. dAgostino, A. Agrestini, and V. Monesi 1974 Nucleolar activity of rat primary spermatocytes. Exp. Cell Res., 86:166-170. Stevens, J.K., T.L. Davis, N. Friedman, and P. Sterling 1980 A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections. Brain Res. Rev., 2:265-293. Tres, I,.L. 1975 Nucleolar RNA synthesis of meiotic prophase spermatocytes in the human testis. Chromosoma, 53;141-151. Uddin, M., G.G. Altmann, and C.P. Leblond 1984 Radioautographic visualization of differences in the pattern of 13HHJuridineand [“Hloroticacid incorporation into the RNA of migrating columnar cells in the rat small intestine. J. Cell Biol., 98:1619-1629. Urena, F., and A.J. Solari 1970 Three dimensional reconstruction of the XY pair during pachytene in the rat (Rattus noruegicusl. C hrornosoma, 30:258-268. Utakoji, T. 1966 Chronology of nucleic acid synthesis in meiosis of the male Chinese hamster. Exp. Cell Res., 42:585-596. Vitate, R., D.W. Fawcett, and M. Dym 1973 The normal development of the blood testis barrier and the effects of clnrniphenc and estrogen treatmcnt. Anat. Hec., 176:333-344.
Nucleolar Structure and Synthetic Activity During Meiotic Prophase and Spermiogenesis in the Rat MICHAEL C. SCHULTZ AND C. P. LEBLOND Department of Anatomy, McCill University, Montreal, Quebec, Canada H3A 2B2
ABSTRACT The ultrastructure of nucleoli was examined in developing rat spermatocytes and spermatids, with the help of serial sections. In addition, the radioautographic reaction of nucleoli was examined in rats sacrificed 1 hr after intratesticular injection of 'H(5')-uridine and taken as an index of the rate of synthesis of ribosomal RNA (rRNA). Primary spermatocytes from preleptotene to zygotene have small nucleoli typically composed of fibrillar centers, a fibrillar component, and a granular component, within which are narrow interstitial spaces. During early and mid-pachytene, nucleoli enlarge to about nine times their initial size, with the fibrillar and granular components forming an extensive network of cords-a nucleolonema-within which are wide interstitial spaces. Meanwhile, there appear structures identical to the granular component but distinct from nucleoli; they are referred to as extrunucleolar granular elements. Finally, from late pachytene to the first maturation division, nucleoli undergo condensation, as shown by contraction of fibrillar centers into small clumps, while fibrillar and granular components condense and segregate from each other, with a gradual decrease in interstitial spaces. In secondary spermatocytes, nucleoli are compact and rather small, while in young spermatids they are also compact and even smaller. Nucleoli disappear in elongating spermatids. In 'H-uridine radioautographs, nucleolar label is weak in young primary spermatocytes, increases progressively during early pachytene, is strong by the end of mid pachytene, but gradually decreases during late pachytene up to the first maturation division. In secondary spermatocytes and spermatids, there is no significant nucleolar label. In conclusion, rRNA synthesis by nucleoli is low in young spermatocytes. During pachytene, while nucleoli enlarge and form a lacy nucleolonema, rRNA synthesis increases gradually to a high level by the end of mid pachytene. However, during the condensation and segregation of nucleolar components occurring from late pachytene onward, the synthesis gradually decreases and disappears. The small, compact spermatids arising from the @ 1990 WILEY-LISS, INC.
second maturation division do not synthesize rRNA. INTRODUCTION
The radical transformations occurring in cytoplasm and nucleus during spermatogenesis have overshadowed the changes taking place in the nucleolus. Yet i t is known that nucleoli are modified in several ways during meiotic prophase and spermiogenesis. In the rat, for example, they gradually enlarge during the pachytene stage (Daoust and Clermont, 1955; Urena and Solari, 1970; Stefanini et al., 1974); while a t the end of this stage, their number decreases (Daoust and Clermont, 1955) and the dense fibrillar component (pars fibrosa) of the nucleolus segregates from the granular component (pars granulosa) (Stefanini et al., 1974; Leblond, 1981). In spermatids, the nucleoli are small, compact structures which eventually disappear altogether (Leblond, 1981). The role of nucleoli is to synthesize the main RNA components of ribosomes, i.e., the 5.8, 18, and 28s ribosomal RNAs (rRNA) (Jordan, 1978). The synthesis of these species may be examined by radioautography soon after 3H-uridine incorporation since the silver grains that appear over the nucleolus are then due to newly formed rRNA (Fakan and Puvion, 1980; Uddin et al., 1984). The first attempts at 3H-uridine radioautography of spermatocyte nucleoli in locusts (Das et al., 1965) and hamsters (Utakoji, 1966) revealed no reaction, suggesting that rRNA transcription was inhibited during male meiotic pro hase Later, however, .: Stefanini et al. (1974) injected ! H-uridine directly into the rat testis and examined rRNA synthesis by LM radioautography. They observed a reaction over spermatocyte nucleoli, with a peak at mid-pachytene, but a long exposure was required. Thus there would be a rather low rate of rRNA synthesis. Soderstrom (1976) examined rRNA synthesis in the rat and concluded that this activity was limited in mid-pachytene cells. Significant rRNA synthesis, however, was reported in mouse spermatocyte nucleoli, except a t late pachytene (Kierszenbaum and Tres, 1974). Finally, a careful study of cultured human spermatocytes (Tres, 1975) led to the conclusion that 3H-uridine incorporation by
Received October 24, 1988. Accepted March 26, 1990. Address reprint requests to Dr. C . P. Lehlond, Department of Anatomy, McGill University, Montreal, Quehec, Canada. Dr. M.C. Schultz's present address is Fred Hutchinson Cancer Research Center, Seattle, WA 98104.
2
M.C. SCHTJLTZ A N I ) C.P. LELILOND
nucleoli was low a t leptotene, reached a peak a t late zygotene, and declined a t late pachytene. In the hope of unifying the scattered information on the behavior of nucleoli during male meiosis and spermiogenesis, their ultrastructure and ’H-uridine radioautography were examined in the rat under in vivo conditions. The meiotic prophase was divided into three phases: 1)preleptotene, leptotene, and zygotene; 2) early and mid-pachytene; and 3) late pachytene, diplotene, and diakinesis. Nucleoli were also examined in secondary spermatocytes and spermatids a t early steps of development. For the ultrastructural study, “random” sections of the testis, that is, sections collected without regard to serial order and orientation, were used initially. Later, “serial” sections of nuclei were prepared to measure the size of nucleoli. These sections also revealed that primary spermatocytes may contain not only nucleoli which have the usual complement of fibrillar centers, fibrillar component, granular component, and interstitial spaces, but also structures composed of a granular component independent of any nucleolus; these structures will be referred to as extranucleolar granular elements. In parallel with the ultrastructural study, the incorporation of 3H-uridine into the nucleolus was assessed in vivo by EM radioautography of random sections. The abundance of silver grains over nucleoli, used as a n index of the rate of rRNA synthesis was related t o the changing features of nucleoli in spermatocytes and spermatids.
outline of each cross section was traced onto transparency sheets, and the tracings were collectively weighed on a Sartorius analytical balance. Since the weight was proportional to the actual volume, this volume was calculated from the final magnification of the micrographs ( x 24,000) and the mean section thickness (60 nm, estimated by direct measurement of re-embedded sections cut a t 90 degrees to the original plane of section). In the case of the largest nucleolus in a given nucleus, not only the total volume, but also that of its granular component was measured by the cut-out weighing method. The volume of the rest of the nucleolus, that is fibrillar centers and fibrillar component, was obtained by subtracting the volume of the granular component from the total volume. Next, the proportion of the granular component occupied by interstitial spaces was determined by the point-hit method (Elias and Hyde, 1983) using a grid lattice containing 56 points, with the dist,ance between points representing three times the average diameter of the ribonucleoprotein granules (RNP) of the granular component. Finally, the volume of each extranucleolar granular element was measured by the cut-out weighing method in serially cut nuclei. ’H-Uridine Radioautography
To examine the incorporation of “H(5’1-uridine into RNA by the nucleoli of spermatocytes and spermatids, adult rats were anesthetized and the ri h t testis was exposed for direct injection of 0.25 mCi of, H(5’buridine MATERIALS AND METHODS (specific activity, 26.0 Ci/mM; New England Nuclear) in Preparation and Analysis of Serial Sections physiological saline. The skin was then sutured, and 1 Testes were obtained from adult Sherman rats h r later the testes were fixed by retrograde perfusion as weighing 190-380 gm. The fixative, 5% glutaralde- above using 2.5% glutaraldehyde. Tissue blocks were hyde in 0.1 M sodium cacodylate buffer at pH 7.3, was then immersed in primary fixative for 2 hr, rinsed, and given by retrograde perfusion into the abdominal aorta postfixed for 2 h r in 1%osmium tetroxide. Processing for after clamping the aorta above the kidney and cutting EM radioautography was done as previously described the renal veins (Vitate et al., 1973). The tissue blocks (Schultz e t al., 19841, using either a 2-month or, in most were then immersed for 1 hour into reduced osmium, cases, a 6-month exposure. Preliminary light-microthat is a 1:l mixture of 2% aqueous osmium tetroxide scopic radioautographs showed widely different radioand 3% potassium ferrocyanide (Karnovsky, 1971). activities in the various parts of the testis. Hence, the The samples were embedded in Epon, and the sections relative labeling intensity over the various cell types were routinely stained with uranyl and lead salts. was assessed subjectively in comparable areas (see TaSerial sections of nuclei were examined in primary ble 3 below). In one case, labeling intensity was quanspermatocytes a t specific stages of the cycle of the se- titated in four adjacent tubules surrounding a large miniferous epithelium, a s classified by the method of blood vessel which presumably provided them with a Leblond and Clermont (1952). The serial sections were similar amount of radioactive precursor. Silver grains mounted on Formvar-coated slotted grids according to were counted over the fibrillar component (with fibrilStevens et al. (1980). Thus one set included 93 consec- lar centers included), whose area was determined by the utive sections showing part of three tubules a t stages I, cut-out weighing method as above. The grain counts VII, and XII, respectively. The profile of each nucleus were expressed per unit area and averaged. under study was photographed in every serial section using a Philips 300 or a 400T electron microscope. In other series mounted on wide-mesh grids, larger sets of sections were obtained, in which a t least every fifth section through a particular nucleus was photographed. In each serially sectioned nucleus, the volume of the largest nucleolus and the largest extranucleolar granAhhrruiations hetorochromatin ular element (identified by surveying the sections) was Chr fibrillar component determined as follows. The total area of a structure in rfc fibrillar center each cross section was determined by the “cut-out g granular component weighing” method (Elias and Hyde, 19831, that is, the NE nuclear envelope
NTJCLEOLUS I N RAT SPP:ItMATOC;ENl(’ CELIA
Fig. 1. Nucleolus in aplaleptolerrr primary spermatocyte (stage VII). The cytoplasm at lower left i s separated by the nuclear envelope from the nucleus, which includes some heterochromatin and, in its center, a nucleolus. Within lhe nucleolus, a light-gray fibrillar center is associated with cords of the fibrillar component. Extending above it is the granular component; i t is formed of patches from which extend short, broad cords; patches and cords arc composed of ribonucleoprotein particles. X 48,500.
3
Fig. 2. Nucleolus in a n eurLy pachytene spermatocyte at stage I. A poorly limited fibrillar center is continuous with cords of the Gbrillar component. The granular component is dispersed in patches (upper right) and slender cords (lower) that form a reticulated network. The interstitial spaces separating the cords of granular component are more lightly stained than the nucleoplasm proper. x 48,500.
Fig. 3.
5
NUCLEOLUS I N RAT SPERMATO(;ENIC CELLS
RESULTS Spermatocyte and Spermatid Nucleoli, As Seen in Random Sections First phase of meiosis
In preleptotene spermatocytes (stage VII of the cycle of the seminiferous epithelium), nucleoli are relatively small (Fig. 1).They include fibrillar centers which appear as pale gray structures, a fibrillar component consisting of few dense cords, and a granular component made up of few patches and cords separated by narrow interstitial spaces. At leptotene and zygotene (stages VIII-XIII), spermatocyte nucleoli show little change in size and appearance. Second phase of meiosis
In early pachytene spermatocytes, i.e., from stage XIV of one cycle to stage IV of the next cycle, there is gradual enlargement of the nucleolus. Fibrillar centers remain small, but the cords of fibrillar component appear more numerous and more loosely organized than in the first phase of meiosis. The granular component enlarges; its patches and cords become numerous, but narrow; they spread out into a tridimensional network, while the interstitial spaces between them widen and appear lighter than the rest of the nucleoplasm (Fig. 2). During mid-pachytene, which extends from stage V to VIII of a cycle, nucleoli occupy a large fraction of the nuclear volume. Fibrillar centers are small, pale-gray structures located at a distance from one another; they remain connected through cords of the fibrillar component. The loosening of fibrillar and granular components noted at early pachytene is enhanced, so that both spread out over a large domain (Fig. 3). In particular, the granular component forms an extensive network composed of numerous small patches interconnected by narrow cords between which are large, light interstitial spaces. Third phase of meiosis
In late pachytene, which extends from stage IX t o XI1 of the cycle, there is a progressive condensation and segregation of nucleolar components. Fibrillar centers change t o compact, electron-dense masses located within an electron-lucent halo (Fig. 4). They are surrounded by dense, thick cords of the fibrillar component which are increasingly separated from the granular component. The patches and cords of the latter agglomerate, leaving only small interstitial spaces between them (Fig. 4). At diplotene (stage XIII) and diahinesis (stage XIV), the condensation of the fibrillar component is accentuated (Fig. 5), and its segregation from the granular component is completed.
Secondary spermatocytes and spermatids
Nucleoli have not been seen during the first maturation division, but reappear in secondary spermatocytes. They are then composed of a fairly solid fibrillar component containing a diffuse fibrillar center, while the granular component condenses on one side of it (not shown). During the second maturation division, nudeoli are again not seen. However, the step 1 spermatids that arise from the final division of spermatogenesis have a small, ovoid nucleolus. It is composed of a distinct, solid fibrillar component containing a diffuse fibrillar center and, as in secondary spermatocytes, the small granular component is segregated from the fibrillar component (Fig. 6 ) .The granular component is lost by the end of step 6 . At step 7, small cavities appear in the fibrillar component and make it difficult to ascertain whether a fibrillar center still exists. At step 8, the nucleolus is exclusively composed of fibrillar component. By step 9, no trace of the nucleolus can be identified. Features of Nucleoli Observed in Serial Sections
Serial sections reveal that, in addition to normal nucleoli in which fibrillar centers and fibrillar and granular components are associated, there may be extranucleolar granular elements (Table 1).The data indicate that, as the cell progresses from leptotene to pachytene, the number of nucleoli decreases, while that of extranucleolar granular elements increases. At late pachytene, however, no extranucleolar granular elements have been observed, except for a single small one. For further analysis, one nucleus has been selected at each stage, and data are recorded for its largest nucleolus and its largest extranucleolar granular element (Table 2). The volume of complete nucleoli increases proqressively from 0.22 km3 at preleptotene to 3.43 km' at late pachytene. Component structures, partic-
TABLE 1. Number of nucleoli and extranucleolar granular elements as observed in serial sections during meiosis' ~
Phase Preleptotene Early pachytene Mid pachytene
~
Approximate No. ExtraNo. of Percent nu c1ear nuclei of nucleus granular examined cut seriallv Nucleoli elements 1 57 7 0 2 64 3 7 4
78 79 79 93
92 Late pachytene ~
Fig. 3. Nucleolus of a mid-pachytene primary spermatocyte (stage V11). A fibrillar center appears homogeneous and well limited. The fibrillar component i s distributed in cords that are associated with the center or intermingle with cords of the granular cumponent. The latter forms a large, reticulated network extending uver most of the micrograph. The patches and cords of the netwurk are separated by pale interstitial spaces. x 34,900.
3
42
59 73
2 2 1 3 2 2
2 3
7 1 6
5 4
0 0 1
'Only the preleptotene nucleus was completely included in the series. It IS estimated that early, mid- and late pachytene nucleoli occupy, respectively, 90%, 75-907'0, and 50-75%) of each nucleus. Hence, except for the prcleptotene nucleus, the numbers in the last two columns are underestimates.
M.C. SCHIILTZ AND C.P. LEHLOND
Fig. 4. Nucleolus of a late pachyime sperrnatocyte (stage XII) containing a condensed fibrillar center outlined by a light space and surrounded by dense cords of the fibrillar component that tend to separate from the granular component. The latter is composed of closely packed patches and cords, with only small interstitial spaces. x 52,500.
lar component (fg) in which some granules are evident. The section by-passed the granular component, of which only the edge is cut tangentially. ~ 5 2 , 5 0 0 .
Fig. 5. Nucleolus in a primary spermatocyte a t diakinesis. A fibrillar center is surrounded by dense, sharply outlined cords ofthe fibril-
Fig. 6. Nucleolus in a step 1 spermatid. This nucleolus is small. It consists of a compact fibrillar component segregated from the granular component. The fibrillar component includes a slightly paler area which is presumed to be a diffuse fibrillar center (arrowheads). x 52,500.
ularly the granular, also increase; the interstitial spaces enlarge up to mid-pachytene and decrease at late pachytene (Table 2). The extranucleolar granular elements at early and mid-pachytene consist of ribonucleoprotein (RNP) granules arranged in loose patches and cords similar to those in the granular component of nucleoli at the same stage, but their volume is highly variable. Thus,
the large extranucleolar granular element (0.80 m3) recorded at early pachytene is larger than the biggest nucleolus in the same nucleus (0.48 +m3) (Table 2). Another one incorporates a round body, as often does the granular component of nucleoli (Schultz et al., 1984). Extranucleolar granular elements may be minute; thus, a small one has been found which is embedded within the condensed XY pair. Finally, at late
NUCLEOI~USIN RAT SPEKMATOGENIC CELIJS
7
TABLE 2. Size of nucleoli and extranucleolar granular elements measured in serial sections in one nucleolus at each phase of the rat meiotic prophase and in a young spermatid'
Prel eptotene Early pachytene Mid pachytene Late pachytene Step 1 spermatid
Stage VII I VII XI1 I
Number of nucleoli Per nucleus 7 3 3
Whole nucleolus 0.22 0.48
1.91
2
3.43
1
0.09
Volumes in largest nucleolus (pm3) Center Granu+ fibrillar lar comcompoponent nent 0.04 0.15 0.15 0.14 0.26 0.64 0.47 2.57 0.06 0.03
Interstitial spaces 0.03 0.19 1.01
0.38 -
Extranucleolar granular elements No. Volume of Per largest one nucleus (pm3 0 7 0.80 5 1.20 0 -
-
'The complete and incomplete nucleoli were from the same nucleus a t each stage, except at mid-pachytene, where they came from two different nuclei.
TABLE 3. Label incorporation by nucleoli and nucleoplasm during meiotic rophase and spermiogenesis, (1 hr) after ..p H-uridine injection into rat testis
Leptotene
Early pachytene Mid-pachytene Diakinesia Earlv mermatid
Staee of cvcle IX XIV,II V1,VII XI11 I
Intensity of labeling' Nucleolus Nucleoolasm +I+ + +
++ ++++ + I0
++ ++-I ++ ++
Over the nucleoplasm, silver grains are present at all stages of meiosis (Figs. 7, 8) as well as in young spermatids (Fig. 9). The reaction increases to a peak at mid pachytene and decreases thereafter, but remains definite in young spermatids (Table 3). DISCUSSION Structural Changes in the Nucleolus of Spermatocytes and Spermatids
The early primary spermatocytes from preloptotene to zygotene had small nucleoli. The limited but precise 'Relative intensity is given in ascending order from 0 (when labeling data in Table 2 indicated that, in the course of early is absent or doubtful) to a maximum of + + i t (when it is intense). and mid pachytene, nucleoli formed an extensive, lacy The reaction over the nucleolus is attributed to newly-formed ribosomal RNA. The reaction over the nucleoplasm is attributed to newly- nucleolonema and thus gradually enlarged to about 9 times their initial volume, due in part to an increase in formed messenger and transfer RNA. the RNP granules (about fourfold) and interstitial spaces (over 30 fold) of the granular component. In the pachytene, the single small extranucleolar granular el- meantime, extranucleolar granular elements of variement (0.05 pm3) is composed, as in nucleoli at that ous sizes made their appearance. Such structures had stage, of a closely packed aggregate of RNP particles. been previously observed in human spermatocytes at leptotene (Stahl et al., 1983; Paniagua et al., 1986), that is at an earlier phase than in the rat. The extraEM Radioautographic Survey of nucleolar granular elements were likely to be frag3H(5')-Uridine Incorporation ments of that granular component that had been In testicular tissue fixed 1 hr after injection of 3H- cleaved from complete nucleoli, presumably as a result uridine, a radioautographic reaction is observed over of the chromosomal movements observed by Parvinen the nucleoli of primary spermatocytes (Table 3). The and Soderstrom (1976) in rat spermatocytes from late reaction is moderate during the first phase of meiosis, leptotene to mid-pachytene. but increases gradually during the second phase, being By late pachytene, the three main nucleolar parts fairly strong at early pachytene and intense through- condensed gradually. Fibrillar centers became particout mid-pachytene up to the end of stage IX (Fig. 7). At ularly dense. The fibrillar and granular elements segeach phase, the reaction is essentially localized over regated, while the interstitial spaces present within the fibrillar component. During late prophase and fol- them decreased markedly. Meanwhile, the extranuclelowing stages (third phase of meiosis), nucleolar reac- olar granular elements disappeared more or less comtivity is markedly reduced. Thus, at diakinesis, there pletely. It was likely that they had rejoined the nucleare rare silver grains over the aggregated cords of the oli, and thus contributed to their enlargement at late fibrillar component and none over other nucleolar pachytene. parts (Fig. 8 ) . In early spermatids, nucleoli are not laNucleoli were small and compact in secondary sperbeled (Fig. 9, Table 3). Some quantitative measure- matocytes and even more so in spermatids1 (Schultz et ments confirmed these conclusions. For instance, in al., 1984);they disappeared by step 8 of spermiogenesis. four adjacent tubules that appeared evenly labeled, counts of the mean number of silver grains per pm2 of fibrillar component were as follows: 44.6 in early pachytene (stages I and 111, 127.3 in mid pachytene from published photographs, compact, segregated nucleoli (stage VII), 4.5 at diakinesis (stage XIV), and a back- are'Judging present in the young spermatids of hamster (Barcellona and Brinkground count in secondary spermatocytes (stage XIV) ley, 1973), mouse (Krimer and Esponda, 1979; Czacker, 1984, 19x5; as well as in step 1 and 2 spermatids (stages I and 11). Mirre and Knibiehler, 1985), and guinea pig (Ohtomo, 1981). 0
Figs. 7-9.
Changes in rRNA Synthesis in Spefmatocytes and Spermatids
The role of the ribosomal genes present in fibrillar centers (reviewed in Perry, 1981; Hugle et al., 1985; Aris and Blobel, 1988) is to produce a primary transcript, the 45s rRNA, which passes into the fibrillar component, while associating with protein. It is then split into 1)a n 18s rRNA which leaves the nucleolus to be built into the minor (405) ribosome subunits, and 2) a 32s rRNA, soon split into 28s and 5.8s rRNA, both of which are presumed to be part of the granular component; they will eventually be built into the major (60s) ribosome subunits. When, a s in the present investigation, radioautographic reactions are observed over the fibrillar component soon after “H-uridine injection, they are attributed to newly formed 45s rRNA and its immediate progeny; and therefore, can be used a s a n index of rRNA synthesis (Fakan and Puvion, 1978; Uddin et al., 1984). The intensity of radioautographic reaction over spermatocyte nucleoli indicated that rRNA synthesis was low from preleptotene to zygotene, gradually increased during early pachytene to a maximum a t midpachytene, and then declined during late pachytene to become negligible a t diplotene. No significant rRNA synthesis occurred in the nucleoli of secondary spermatocytes and spermatids. Incidentally, the intensity of silver staining by nucleoli which, in the past, had been taken as a n index of rRNA synthesis was more likely to indicate the presence of particular proteins (Goessens, 1984). Thus, even though spermatid nucleoli were stained with silver (Hofgartner et al., 1979), they showed no evidence of rRNA synthesis. Interprefation of the Results
When nucleoli were examined in various types of differentiating cells (blood cells-Busch and Smetana, 1970; keratinocytes and sebaceous cells-Karasek et al., 1972, 1973; epithelial cells in alimentary tractAltmann and Leblond, 1982; Lee, 1985), it was con-
~~
~~
Fig. 7. Radioautugraph of the nucleolus from a spermatocyte ob-
served at about the end of mid-pachytene (stage 1x1 fixed I hr after intratesticular injection of “H-uridine. Fibrillar and granular components are prominent, hut there is some decrease in size of the interstitial spaces. Radioautographic silver grains are numerous over the fibrillar component; while over the granular component grains are rare, except close to the fibrillar component. There is a fair reaction throughout the nucleoplasm. XY, Condensed sex pair. x 30,000. Fig. 8. Radioautograph of the nucleolus from a primary spermatocyte at diakrnesis (stage XIVi 1 hr after %IH-uridineinjection. The fibrillar component which includes compact fibrillar cunters (arrows) is segregated from the condensed granular component which surrounds a “round body” (KR) with a large vacuole in thc center. Silver grains are rare over the fibrillar component and may be due to background fog. No silver grains arc present over the granular component. The reaction over the nucleoplasm is moderate. x 20,400. Fig. 9. Radioautograph of the nucleoluv from a step 1 spermatid 1 hr after 3H-uridine injection. The fibrillar component encloses a faintly visible fibrillar center (arrowheads) and a large round body (RBI. The scanty granular component includes only a few RNP particles. No silver grains are seen on any part of the nucleolus. However, thcre is a moderate reaction in the nucleoplasm. x 48,000.
cluded that undifferentiated proliferative cells had active nucleoli, characterized by a large, highly reticulated network, the nucleolonema, whereas fully differentiated cells usually had less active or inactive nucleoli, which were often small and compact. For example in r a t small intestine, a s columnar cells differentiated during their migration from crypt base to villus top, nucleolar volume decreased by a factor of 16, while fibrillar and granular components gradually condensed and became segregated (Altmann and Leblond, 1982). In the meantime, counts of silver grains over nucleoli in 3H-uridine radioautographs were high in crypt base, gradually decreased during the migration from crypt to villus, and were a t background level in villus top (Uddin et al., 1984). Thus, in general, the rRNA synthetic rate decreased in parallel with nucleolar size. During early and mid-pachytene, nucleolar volume increased and a nucleolonema-like network developed, while rRNA synthesis increased markedly. These findings were indicative of a n activation of ribosomal gene transcription, in contrast to the previous view that rRNA synthesis was lacking (Das et al., 1965; Utakoji, 1966) or low (Stefanini et al., 1974) during male meiosis. The ribosomal gene activation during early and mid-pachytene was associated with a fourfold increase in RNP granules (Table 2). In the case of somatic mitosis, only a doubling of these granules was observed during the cycle (Lepoint and Goessens, 1982). The next phase of spermatocyte development, extending from late pachytene to the first maturation division, was characterized by a decrease in radioautographic reaction, which fell to a negligible level during diplotene. Meanwhile, there was condensation and segregation of nucleolar components, but no decrease in nucleolar size, or rather a n increase, attributed in part to aggregation of extranucleolar granular elements as mentioned earlier and in part to the possible fusion of whole nucleoli (as suggested by the figures in Daoust and CLermont, 1955). Nevertheless, the waning of rRNA synthesis and the segregation of nucleolar components demonstrated the inactivation of ribosomal gene transcription. The shutdown was similar to t h a t observed at metaphase and anaphase in the mitotic cycle of somatic cells (Fan and Penman, 1971; Lepoint and Goessens, 1978). Nucleolar rRNA synthesis was promptly restored at the end of somatic mitoses, but this was not the case in the secondary spermatocytes arising from the first maturation division nor in the spermatids arising from the second. The level of rRNA synthesis in spermatids had been previously examined by biochemical studies of spermatids separated by centrifugation. In contrast to our observations, Geremia et al. (1978) reported that “quasi-homogeneous” preparations of round spermatids from mouse tubules labeled with ,’H-uridine in vitro synthesized as much rRNA as mid-late pachytene spermatocytes. However, Meistrich e t al. (1981) isolated 985% pure pachytene spermatocytes and 93% pure round spermatids after intratesticular injection of H-uridine to rats and found that pachytene cells were active in rRNA synthesis, but round spermatids were not, in agreement with our findings. Finally, Kierszenbaum and Tres (1974, their Fig. 9) depicted in spermatid nuclei a dense spherical component which showed
10
M.C. SCHIJLTZ AN11 r.1’.LEKLONI)
no reaction in 3H-uridine radioautographs; our identification of this component as a nucleolus was in accord with the conclusion that spermatid nucleoli did not synthesize rRNA. In conclusion, the activation of ribosomal genes in nucleoli during early and mid pachytene is followed by inactivation just before the two meiotic divisions take place. Unlike what happens after somatic mitosis, there is no significant reactivation of ribosomal genes in the young spermatids arising from meiosis, ACKNOWLEDGMENTS
The first author successively received a David Stewart Memorial Fellowship from McGill University and a Foundation “ravelling Scholarship from the University of Queensland, Australia. Funding for the project was provided through a grant from the Medical Research Council of Canada. Critical reading of the manuscript by Dr. Y. Clermont is gratefully acknowledged. Ms. Sonia Bujold is thanked for her excellent technical assistance throughout the course of the work reported in this and the companion article. LITERATURE CITED Altmann, G.G., and C.P. Leblond 1982 Changes in the size and structure of the nucleolus of columnar cells during their migration from crypt base to villus top in ratjejunum. J. Cell Sci., 66.83-99, Aris, d.P., and G. Blobel 1988 ldentification and characterization of a yeast nucleolar protein that is similar to a rat liver nucleolar protein. J. Cell Biol., 107~17-31. Barcellona, W.J., and B.R. Brinkley 1973 Effects of actinomycin D on spermatogenesis if the Chinese hamster. Biol. Reprod., 13:335349. Busch, H., and K. Smetana 1970 The Nucleus. Academic Press, New York. Czaker, R. 1984 Observations on the dynamics of argyrophilic nucleolar material in the nuclei of mice spermatids. .Experientia, 40: 960-963. Czaker, R. 1985 Ultrastructural observations on nucleolar changes during mouse spermiogenesis. Andrologia, 17:42-53. Daoust, R., and Y. Clermont 1955 Distribution of nucleic acids in germ cells during the cycle of the seminiferous cpithelium. Am. J. Anat., 96:255-283. Das, N.K., E.P. Siegel, and M. Alfert 1965 Synthetic activities during spermatogenesis in the locust. J. Cell Hiol., 25:387-395. Elias, H., and U.M. Hyde 1983 A Guide to Practical Stereology. S. Karger, Basel. Fakan, S., and E. Puvion 1980 The ultrastructural visualization of nuclcolar and extranucleolar RNA synthesis and distribution. Int. Rev. Cytol., 65:255-299. Fan, H., and S. Penman 1971 Regulation of synthesis and processing of nucleolar components in metaphase-arrested cells. J. Mol. Biol., 5Y:27-42. Geremia, R., A. D’Agostinm, and V. Monesi 1978 Biochemical evidence of haploid gene activity in spermatogenesis of the mouse. Exp. Cell tles., lIlt23-:30. Goessens, G. 1984 Nucleolar structure. Int. Rev. Cytol., R7;107-158. Hofgartner, F.J., M. Schmid, W. Krone, M . T ~Zenses, and W. Engel 1979 Pattern of activity of nucleolus organizers during spermatogenesis in mammals as analysed by silver-staining. Chi-omosoma, 71~197-216. Hiigle, B., R. Hazan, U. Scheer, and W.W. Franke 1985 Localization of ribosomal protein S1 in the granular component ofthe interphase nucleolus and its distribution during mitosis. J. Cell Biol., 100: 873-886. Jordan, E.G. 1978 The Nucleolus. Carolina Biology Readers, Burlington, North Carolina. Karasak, J., K. Smetana, A. Hrdlicka, T. Dubinin, 0. Hornak, and W. Ochlert 1972 Nuclear and nucleolus ultrastructure during the
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