Sequential changes in the nucleoli of human spermatogonia with special reference to rDNA location and transcription

TISSUE 0

AND

1090 Longman

CELL,

IYYO 22 (1)

Group

UK

25-37

Ltd

M. HARTUNG*,

F. WACHTLERt, A. de LANVERSIN”, C. FOUET*, H. G. SCHWARZACHERt and A. STAHL*

SEQUENTIAL CHANGES IN THE NUCLEOLI OF HUMAN SPERMATOGONIA WITH SPECIAL REFERENCE TO rDNA LOCATION AND TRANSCRIPTION Keywords:

Man,

ABSTRACT. silver

spcrmatogonia.

The

staining,

nucleoli

were consistently associated

in the

nuclei

morphology The

and

showed

was found observed

of rDNA

consistently

found

the

nucleoli.

did wcrc

in fibrillar

deposit

reflect

ccntcrs

was often

with

located

indicator

except

in the

sltc of the

radioautographs.

of spermatogo-

uptake.

tibrillar

in the dcnac fihrillar

and nontranscribed

threads. portmns

was with

demonstrated center

clearly

component.

component.

cc\sa-

the label

B spcrmatogonia

this finding

at the cdgc of the tihrillar

and transcrlhed

of spermatogomal

01 siru hybridization

to the

located

Nucleolar

to a prc-lcptotcnc

‘H-uridinc

and in the dense fihrillar

localized

proteins

to disintegrate.

corresponds

hc detected.

correspond

a single fihrillar

were centrally

the differentiation

following

component.

could

not

Compared

units of rDNA

silver-stained

tihrillar

no uptake

of rDNA

positive

nia the silver between

dense

and contained

found

microscopy.

of A spermatogonia,

nucleoli

not an unequivocal

In radioautographs

component.

transcrihcd

was strongly

the

or were

morphology

using electron

In all types

accn in B spcrmatogoma

where

distribution

the dense fihrillar that

over

studied

of the nucleus

centers

though

in nuclcolar

disintegratmn

were

In B spcrmatogonia.

fihrillar

to hc a good,

changes

gcncs

spcrmatogonia

at the pcriphcry

acveral

transcription.

disintegrating that

located

rihosomal

in siru hybridization.

and

with the nuclcarcnvelope.

nia: the nuclcolar tion

of human

radioautography

center

type.

nucleoli

nucleolus.

hut

to

cstahlished

Silver

staining

In Ap spcrmatogoThe

rclationshipb

of rihosomal

genes

arc reevaluated.

Introduction On the basis of cytological characteristics, three or four main types have been identified in the human spermatogonial population but their sequential development and differentiation is still controversial. Nucleolar morphology is one of the criteria by which different classes of spermatogonia have been characterized. According to Clermont (1963,1966), three main spermatogonial types can be identified using light microscopy: the dark type A spermatogonia (Ad), considered as the stem cell, the pale A spermatogonia (Ap) and the type B spermatogonia, these being more *Lahoratoire boulevard

de GCnCtique,

Jean-Moulin.

+Histologisch-Embryologisches Wien,

Austria.

Received

26 July 1989.

FacultC

de MCdecinc.

13385 Marseille Institut

Ccdex

27.

5. France.

der UniversitPt

differentiated ceils giving rise to preleptotene spermatocytes. A fourth type, the long type A spermatogonium (Al), has been described in the electron microscope by Rowley et al. (1971). as the germinal stem ceil. These statements have been questioned by Chowdury et al. (1975), W. Shultze (1978) and C. Schultze (1979). However, quantitative data obtained by Paniaga et al. (1987) suggest that Ad spermatogonia are the stem cells, as previously postulated by Clermont. Although the location and the shape of the nucleolus belong to the main morphological features used to distinguish the four classes of spermatogonia (Rowley et al., 1971; Paniaga et al., 1986), the nucleolar aspects have not been thoroughly studied. An understanding of the nucleolar morphology is, however, essential for the identification of these cells in electron microscopy. The

26

HARTUNG

peripheral placement of the nucleolus in Al, Ad and Ap spermatogonia and its central position in the nucleus of B spermatogonia have been stressed, but the spatial distribution of the nucleolar components (fibrillar center, dense fibrillar component, granular component) has not been systematically investigated. The disparate and fragmentary descriptions of nucleoli in spermatogonia prompted us to reinvestigate this organelle in spermatogonia. The localization of ribosomal genes in nucleoli and the meaning of silver-staining are a matter of controversy. The relationship between fibrillar centers and transcription is unclear. It has been suggested that the fibrillar centers correspond to the non-transcribed part of rDNA in the nucleolus organizer (review in Hadjiolov, 1984) and that the surrounding dense fibrillar component is the site of transcription of ribosomal genes. The demonstration that fibrillar centers contain RNA polymerase I (Scheer and Rose, 1984; Raska et al., 1989) may bring in question this interpretation. In the present study, we used electron microscopy, high resolution radioautography following ‘H-uridine uptake, the NOR-silver technique in light and electron microscopy and in situ hybridization. The different types of spermatogonia were identified according to the criteria described by Holstein and Roosen-Runge (1981). We report here the morphological characteristics of nucleoli, the sites of rDNA transcription and the distribution of silverstained proteins in the different types of spermatogonia. The distribution of ribosomal genes could be correlated to the nucleolar components by in situ hybridization in Ap spermatogonia. Moreover. we found that nucleoli undergo breaking up and disappear in B spermatogonia when they have reached the preleptotene stage, and are formed de nova in leptotene spermatocytes. Materials and Methods Testicular samples The testicular samples originated from five patients that underwent orchidectomy as a part of their treatment for prostatic carcinoma. Their testicular histology was normal. The patients had received no radiation, hormone treatment, or chemotherapy prior to surgery.

ETAL.

Electron microscopy Testicular biopsies were fixed in 2.5 glutaraldehyde in 0.1 M phosphate buffer containing 0.05 M sucrose, pH 7.2, at 4°C followed by 2% osmium tetroxide in the same buffer. After dehydration in a graded series of acetone, the specimens were embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and examined in a Jeol 100 electron microscope. Silver staining at the ultrastructural

level

Silver staining was carried out according to Hernandez-Verdun et al. (1980). The silverstained grids were stained with uranylacetate in addition to the silver stain. High resolution radioautograph) Small testicular pieces (1 mm’) were incubated for 30min at 25°C in 1 ml of 80%’ Eagle’s minimal medium/20% calf serum containing 100 &i/ml “H-uridine (specific activity 24 CiimM, CEA France). After fixation and embedding, the sections were transferred onto collodion coated slides and treated according to the dipping method (Salpeter and Bachman, 1972). For coating. an Ilford L4 emulsion (dilution I :4) was used. After exposure for 2 months, the preparations were developed in Mikrodol-X (Kodak) and fixed using Kodak rapid fixer. The sections were stripped off the glass slides onto a water surface, transferred onto grids and stained with uranyl acetate and lead citrate. Preparution microscopy

of’ testicular

samples fbr light

Small pieces of testicular material were immersed in a cold mixture oi methanol and glacial acetic acid (3: 1: v/v) for 10 min. After that, material was brought into suspension in 45% glacial acetic acid in distilled water. This suspension was dropped onto glass slides and air-dried. Silver-stuining and removal of’ silver staining Silver-staining was carried out according to Bloom and Goodpasture (1976). Pictures were taken from suitable nuclei. Subsequently silver was removed by treating the preparations with a solution containing 1% sodium thiosulphate and 1% potassium ferricyanide in distilled water for 20 min. After

HUMAN

21

SPERMATOGONIA

that, the slides were thoroughly rinsed in distilled water and dehydrated in graded ethanol series. In situ hybridization using a nonradioautographicprobe The rDNA probe is a biotinylated 5.2 kB BglII-EcoR1 fragment (for details of the probe and pretreatment of the preparations with RNAse A and proteinase K see Wachtler et al., 1989): 10 ~1 of a mixture containing 20 ng/pl of the probe, 1 pgI/.d salmon sperm DNA, 1 CLgIpl yeast RNA and 10% dextran sulfate (Sigma, Mol. Wt. 500,000) in 50% deionized formamide in 2 x SSC were placed onto slides. Denaturation of the probe and the specimens was done simultaneously by putting the slides covered with coverslips into an incubator at 90°C for ten min. After that, the slides were transferred into moist chambers and these into an incubator at 37°C and left there overnight. Detection of the probe with FITC-labelled Avidin (Vector) was performed according to Pinkel et al., 1986 with some modifications (Wachtler et al., 1989). As controls, metaphase preparations of human chromosomes obtained from human PHA-stimulated lymphocytes were processed together with each batch of testicular samples. In metaphases, signals were observed only in the secondary constrictions of the human acrocentric chromosomes. Additionally, chromosome preparations were hybridized following the same protocol as given above, but without a DNA probe or with biotinylated plasmid DNA of similar GC-content as human rDNA (pGEM, PROGEMA) instead of the rDNA probe. In this case no signal could be detected over nuclei or chromosomes. Results A dark spermatogonium

(Ad)

The nucleus of the Ad spermatogonia contained a vacuole filled with a fine fibrillar matrix and irregular granules. The nucleoli were attached to the nuclear envelope (Fig. 1). They displayed a lenticular shape, their nucleoplasmic side being convex. The single fibrillar center was connected to the lamina densa of the nuclear envelope. Its shape was rounded, but sometimes irregular. The fibrillar center was incompletely surrounded by

threads of dense fibrils that extended away from the fibrillar center and were separated by clear interstices. The nucleolar periphery close to the nucleoplasm was granulo-fibrillar or granular. Silver-staining was positive in the fibrillar center and in some areas of the dense fibrillar component (Fig. 2). On radioautographs, the label was seen overlaying the dense fibrillar component and the fibrillo-granular threads (Fig. 3). A long spermatogonium

(Al)

The nucleoli were consistently located at the nuclear envelope. Different types were observed: (1) Nucleoli with a large fibrillar center, surrounded by an irregular sheet of dense fibrils, and a small granular component (Fig. 4), or (2) large round nucleoli, with a medium-sized fibrillar center surrounded by the fibrillar component. This component was continuous with nucleolonemal granulofibrillar threads, showing electron-translucent interstices. The fibrillar center was always associated with the lamina densa of the nuclear envelope (Fig. 4). The fibrillar center and the dense fibrillar component were found to be stained with silver (Fig. 5). After a ‘H-uridine pulse, the label was seen only over the dense fibrillar component and the fibrillo-granular threads (Fig. 6). A pale spermatogonium

(Ap)

The most polymorphic nucleolar types were seen in this class of spermatogonia. However, association with the nuclear envelope was a consistent feature. A first type was represented by nucleoli with a compacted nucleolonema and small interstices. The single and large fibrillar center was incompletely surrounded by the dense fibrillar component. The latter extended towards the nucleoplasm with anastomosing fibrillo-granular sheets forming a convex protrusion (Fig. 7). The second type displayed a single and relatively small fibrillar center attached to the lumina densa and a loose reticular nucleolonema the proximal part of which was formed by dense fibrils, while the distal part, extending into the nucleoplasm, was fibrillo-granular. The nucleolonemal part of the nucleolus was large and protruded towards the central region of the nucleus (Fig. 8) In a third type, the fibrillar center was also attached to the lamina densa. Irregular

-_._

-

..

-._

___

HUMAN SPERMATOGONIA

29

threads of dense fibrils were closely attached to the fibrillar center. The nucleolonemal threads appeared scattered, separated by interstices containing a nucleoplasm-like material. The surface of the nucleolonema was irregular and showed thread-like, mainly granular, projections in the nucleoplasm (Fig. 9). Silver impregnation stained both the fibrillar center and the dense fibrillar component (Figs. l&12). In the compacted type, the large fibrillar center was heavily stained (Fig. lOa,b). The dense fibrillar component showed silver deposits either in a random location, or at the edge of the anastomosing cords (Fig. lob). The peripheral silverstaining of the anastomosing threads or sheets was an almost constant feature of the reticular type, mainly in the dense fibrillar areas (Fig. lla, b). The silver reaction was also positive in the nucleoli with irregular outline and thread-like projections, where the fibrillar center and the dense fibrillar component were stained (Fig. 12). Among 60 nucleoli studied with radioautography following “H-uridine uptake, only 4 showed one silver grain overlaying the fibrillar center. In contrast, labelling was consistently observed over the dense fibrillar component and the fibrillo-granular threads. In some nucleoli of the nucleolonemal type that were very large, the label extended far away from the fibrillar center (Fig. 13). In the third type of nucleoli, where the nucleolonema was disrupted, the grains were loca-

lized mainly on the dense fibrillar component close to the fibrillar center. In situ hybridization Prior to hybridization, the nucleoli were stained with silver. In the light microscope, the fibrillar center appeared as a small, round, black area whereas the other nucleolar components were less heavily stained. Type A spermatogonia were identified by their displaying 4-6 nucleoli, most of them located in the peripheral region of the nucleus. After removal of the silver deposit, hybridization was performed. Subsequent fluorescent visualization of the probe never revealed a single round area as would be expected if rDNA were confined to the fibrillar center. Comparison of silver-staining and in situ hybridization showed that fluorescence extended beyond the fibrillar center into a nucleolar area that corresponds to the parts of the nucleolonema composed of dense fibrils and of fibrillo-granular threads as shown by electron microscopy (Fig. 21a, b). Nucleologenesis at telophase

Mitotic figures were frequently found, but it was not possible to determine to which type of spermatogonia they belonged. At early telophase, prenucleolar structures were observed. They consisted of small roundish structures located at the periphery of the chromosomes (Fig. 14). They had the same fibrillar appearance and low electron density as fibrillar centers. The prenucleolar struc-

Figs 1-3. Nucleoli in A dark spermatogonia. Fig. 1. The fibrillar center (FC), attached to the nuclear envelope. is partially surrounded by the dense fibrillar component (DFC). The peripheral areas arc mainly granular. x 19,OW. Fig. 2. Silver staining. The FC and some areas of the DFC arc silver-positive. ~24.000. Fig. 3. Radioautography following ‘H-uridine uptake: labelling grains are at the periphery of the FC, mainly overlying the DFC. x24,OW. Fig. 4-6. Nucleoli in A long spermatogonia Fig. 4. The large and small FCs are attached to the nuclear envelope. x28.000 Fig. 5. Silver staining. The FC and the DFC arc stained with silver. ~30.OtXl Fig. 6. Radioautography following ‘H-uridine DFC. ~31,000. FC: Fibrillar center. V: vacuole.

uptake. The label is mainly seen over the

0‘0 a ‘f’ i

j.

-_

.

HUMAN

31

SPERMATOCONIA

tures were similar to those described in human TG cells (Hernandez-Verdun et al., 1980), in human cancer cell lines and in murine Ehrlich cells (Ploton et al., 1987), except for the dense fibrillar component that was not yet differentiated at this stage.

labelling over the dense fibrillar threads (Fig. 17). 2. In the second type, the nucleolar components were disintegrated. Fibrillar centers and dense fibrillar component had disappeared. The fibrillo-granular or granular strands were dispersed in the nucleoplasm (Fig. 18). Absence of fibrillar centers and of dense fibrillar component suggested that transcription had ceased and that the scattered nucleolar structures were inactive remnants. Absence of transcription was shown indeed by lack of labelling over these residual structures (Fig. 19).

B Spermatogonia

These cells were easily identified since the contact with the basal lamina of the seminiferous tubules was reduced to a small area and the nucleoli had detached from the nuclear envelope and were centrally located. Two nucleolar aspects, differing in their morphology and functional significance, were found in B spermatogonia. 1. In the first type, the nucleoli had a nucleolonemal organization (Fig. 15). They displayed several fibrillar centers incompletely surrounded by dense fibrillar component. The nucleolonemal cords contained fibrils and granules. In some nuclei, chromatin clumps were associated with the nudeolus. The silver reaction was positive in the fibrillar centers and the dense fibrillar component (Fig. 16). Radioautography showed intense

Figs 7-13. Figs 7-9.

Nucleoli in A Different

with

Fig. 8. Reticulated Fig. 9. Irregular attached

Figs l&12. Fig.

10a.

patespermarogonin

aspects of nucleoli

Fig, 7. Nucleolus

tently

compacted

nucleolus nucleolus

to the lamina Silver

In

Formation of new nucleoli at leptotene

The leptotene stage was characterized by individualization of chromosomes. They showed a single axial core uniformly surrounded by chromatin. The telomeres were attached to the inner membrane of the nuclear envelope. New nucleoli were formed, associated with the secondary constriction of acrocentric chromosomes. They consisted of a fibrillar center partially sur-

the

nucleolonema

with small FC.

with dissociated

and large FC.

x33,ooO

X28,OOO nucleolonemal

densa of the nuclear

threads.

x 13,000.

The FC is consis-

envelope.

staining compacted

type,

the

large

FC

and

parts

of the

DFC

are silver-positive.

x33,cGO. Fig. lob.

Silver

Fig.

and

tibrillar

lla

deposit b. In

the

reticulated

nucleolus,

areas of the nucleolonemal

a, X29,000;

threads,

threads.

the

FC

the silver

x30,000

is stained

deposit

with

displays

silver.

In the

a peripheral

dense

location;

b, x20$00.

Fig.

12. FC and irregular

Fig.

13. Radioautography

ing the dense fibrillar Fig.

at the edge of nucleolonemal

14. Early

and low electron

patches

following

component

telophase: density

of the thread-like ‘H-uridine

and fibrillo-granular

prenucleolar

as FCs.

X 12.ooO.

structures

projections

uptake.

are silver-positive.

The labelbng

areas. (arrows)

X 17,Mw).

grains are seen overlay-

x21 BOO. showing

the same librillar

aspecsts

’

“

HUMAN SPERMATOGONlA

Fig. 21. Comparison of silver staining and the location of rDNA. (a) Nucleus of a spcrmatogonium A as seen after silver staining. The fihrillar centers can he seen as intensely stained structures (arrowheads) within the somewhat less intensely stained nucleoli. (b) Nucleus of the same spermatogonium as in Fig. Zla after removal of the silver staining and subsequwt in situ hybridization. Arrowheads indicate the position of the Abrillar ccntcrs (same positlon as in Fig. 21a). The location of the Ruorescent signal (location of rDNA) does not correspond to the location of the librillar cumxs. x 3ooO.

rounded by a layer of dense fibrils. The fibrillar center was penetrated by chromatin fibers from the secondary constriction. In some nucleoli, the layer of dense fibrils was continuous with fibrillo-granular strands that extended into the nucleoplasm (Fig. 20). emanating

Figs E-19. B. spermatopnia. located.

Discussion Nucleolar morphology in different types of Verm@?oflia Our results indicate that nucleolar structure is not an absolutely reliable criterion for dis-

The nucleoli, detached fromthenuclearenvelope,

arecentrally

Fig. 15. The reticulated nucleoli display several FCs (arrows) partially surrounded by DFC x22,txKl. Fig. 16. Silver stains FCs and arcas of DFC. x22,OCO Fig. 17. Radioautography following “Wuridine uptake. The label is seen at the pcnphery of the FC and over the DFC The granular component (G) is unlahelled. x 19.lMMl Rg. 18. Disintegration of the nucleolar components which are dtspened m the nucleoplasm. FCs have disappeared. X lY,ooO. Fig. 19. Radioautograph followmg ‘H-uridme uptake. showing lack of labelhng over the scattered nucleolarcomponents. x28,OW. Fig. 20. Spermatocyte at leplotene. Detail of the mxIcus, showing two newly formed small nucleoli. composed of a FC partially surrounded by DFC. One of them is associared with the secondary constriction of anuclcolar chromosome displaying a bingleaxialcorc (arrowhead). x25,oMl.

31

tinguishing spermatogonia, since nucleoli with compacted nucleolonema observed in Ad and Al spermatogonia, were also seen in Ap spermatogonia. However, some features are highly characteristic for A and B spermatogonia, respectively. All types of A spermatogonia displayed a single fibrillar center associated with the nuclear envelope. The largest nucleoli, displaying intense transcriptional activity, were seen in Ap spermatogonia. In B spermatogonia, nucleoli were no longer associated with the nuclear envelope and were located in a central nuclear area. Moreover, these nucleoli contained several fibrillar centers. Sequence of nucleolar changes during the differentiation of spermatogonia In all types of Aspermatogonia nucleoli associated with the nuclear envelope were seen. These nucleoli were found to be transcriptionally active. In type B spermatogonia two types of cell, with respect to their nucleolar morphology, could be distinguished: the first was characterized by an active nucleolus in terms of rDNA transcription. Obviously, this type represents an early substage in type B spermatogonia. The second type displayed transcriptional inactivity and a breaking up of the nucleoli. The loss of dense fibrillar component in the nucleolar remnants was similar to that observed after inhibition of rDNA transcription by microinjected antibodies to RNA polymerase I (Benavente et al., 1988). The B spermatogonia that contained only residual nucleolar structures had probably reached the preleptotene stage described by Clermont (1963), where premeiotic DNA synthesis occurs. This second type therefore corresponds to a late substage in type B spermatogonia. The disappearance of nucleoli in preleptotene requires that new nucleoli are formed at leptotene, the spermatocyte stage that succeeds preleptotene without interposition of mitosis. We could confirm that nucleoli are newly formed in leptotene spermatocytes at the secondary constriction region of nucleolar chromosomes, as already mentioned (Stahl et al.. 1983). Transcription of rDNA in nucleoli The distribution and transcription of rDNA in nucleoli is still controversial. It has been

HARTUNG

ETAL.

suggested that fibrillar centers are the interphasic counterparts of NORs, but it has been generally accepted that transcription of rDNA occurs in the dense fibrillar component that surrounds the fibrillar center (review in Stahl, 1982; Schwarzacher and Wachtler, 1983; Goessens. 1984; Hadjiolov, 1984). Surprisingly, RNA polymerase I has been detected in the fibrillar centers and not in the dense fibrillar component, a finding that led Scheer and Rose (1984), Scheer and Raska (1986) and Thiry et al. (1988) to conclude that rDNA is localized and transcribed in the fibrillar center. However, in situ hybridization revealed that rDNA is located in the dense fibrillar component in human Sertoli cell nucleoli, and is not detectable in the fibrillar centers (Wachtler et al., 1989). Since Ap spermatogonia often contain large nucleoli where the single fibrillar center is attached to the nuclear envelope and are thus separated from the other components, these cells are a useful model for studying the location of rDNA in the light microscope. In situ hybridization demonstrated that the distribution of rDNA did not correspond to the site of the fibrillar center which was revealed by silver staining on the same cell. Moreover, radioautography showed that ‘Huridine uptake took place only in the nucleolonemal threads that contain the dense fibrillar component. or in the peripheral region of the fibrillar center. These findings exclude the possibility that rDNA transcription occurs in the fibrillar center proper. They confirm former studies which localize the site of rDNA transcription in the dense fibrillar component. Silver staining reevaluated Silver staining was positive in both the fibrillar centers and the dense fibrillar component in nucleoli from all spermatogonial types. confirming results already obtained in other cells by Hernandez-Verdun (1980). Pebusque and Seite (1981) and Goessens and Lepoint (1982). Since both fibrillar centers and the dense fibrillar component can be stained with silver, but only the dense fibrillar component contains ribosomal DNA, silver staining cannot be taken as a banaJide indicator for transcription. Biochemical and immunolocalization studies have shown that the silver-stained proteins correspond to

HUMAN

SPERMATOGONIA

phosphoprotein C 23 (Ochs and Busch, 1984) also called nucleolin. This 713-amino acid protein, recently sequenced by Lapeyre et al. (1987), is the main silver-staining protein of nucleoli. The dense fibrillar regions contain the highest concentration of protein C 23 (Lischwe et al., 1981; Spector et al., 1984; Escande et al., 1985). By cross-linking with ultra-violet light and precipitation by anti-C 23 antibody, Herrera and Olson (1986) have demonstrated the close association of protein C 23 with nascent pre-rRNA transcripts. Furthermore, protein C 23 binds DNA with a higher affinity for the nontranscribed rDNA spacer than for the transcribed portion of the ribosomal gene (Olson et al., 1983). The key role of nucleolin (C 23) in ribosomal biogenesis, in the structuring of chromatin in the vicinity of ribosomal genes and in nucleolar organization (review in Jordan, 1987) might explain that it is detected by silver staining in nucleolar compartments directly (or indirectly) involved in rRNA transcription. In this respect the peripheral distribution of silver staining in dense fibrillar threads, in Ap spermatogonia, deserves attention. Ploton et al. (1983) observed the same peripheral silver staining of nucleolar cords in breast carcinoma cells, coinciding with localization of DNA, and concluded that the edge of the nucleolar cords might be the site of nucleolar transcriptional activity. The localization of the silver stained protein in both fibrillar centers and dense fibrillar component might be explained by the loop organization of rDNA in Xenopus laevis described by Marilley and Gassend-Bonnet (1989). In this model, gathered rDNA loops may behave as a confinement space for RNA polymerase I. If rDNA loops are located in the dense fibrillar component, in nucleoli where the dense fibrillar component surrounds the fibrillar center, this may be the site of concentration of RNA polymerase I after being released at the termination site of* transcription. This hypothesis could explain the presence of RNA polymerase I

35

within the fibrillar center (Scheer and Rose, 1984; Raska et al., 1989). Considering that the nontranscribed spacer is much longer in man (31 Kb) than in Xenopus (5-10 Kb), it may be located in the fibrillar center. The preferential binding of nucleolin (C 23) to the spacer may account for the silver staining of this nucleolar compartment. Alternatively, the silver-staining of the fibrillar center may correspond to the accumulation of RNA polymerase I molecules in a confinement compartment, since Williams et al. (1982) identified two silver-stained polypeptides in purified nucleoli, which could correspond to RNA polymerase I subunits. If the active rDNA loops are contained in the dense fibrillar component that surrounds the fibrillar center and extends into the nucleolonemal threads in large active nucleoli, the binding of nucleolin to nascent pre-rRNA is the most likely explanation for silver deposit in the dense fibrillar component. In nucleoli where the dense fibrillar component is not limited to the layer surrounding the fibrillar center, the latter could be considered as a structure from which chromatin containing rDNA uncoils for active transcription in the dense fibrillar regions of the nucleolonema. If this interpretation were correct, the fibrillar center would contain the nontranscribed spacer while the nucleolonema1 dense fibrillar cords would contain both transcribed and nontranscribed portions of ribosomal genes. This model, already proposed by Mirre and Stahl (1981) and Stahl (1982), is consistent with the finding that the dense fibrillar component displays the highest concentration of topoisomerase I (Raska et al., 1989) and is in accordance with the results reported in this study. Acknowledgements The authors thank C. Cataldo (CNRS) and M. Soler (CNRS) for technical assistance and Mrs L. Laurens for typing the manuscript. This work was supported by CNRS (U.A. 1189).

Benaventc. R., Reimer, G., Row, K. M., Hiigle-DGrr, B. and Schcer. U. 1988. Nuclcolarchangesaftermicroinjection of antibodies to RNA polymerasc 1 into the nucleus of mammalian cells. Chromosoma, !47, 1 l-%123. Chowdury, A. K., Steinberger. A. and Steinberger. E. 1975. A quantitative study of spermatogonial population in organ culture of human testis. Andrologia. 7.297-307. Clcrmont. Y. 1963. The cycle of the seminiferous cpithclium in man. Am. J. Anar.. 112,3.5-51.

36

HARTUNG

ETAL.

Clermonl, Y. 1966. Renewal of spermatogonia m man. Amer. J. Arm., 118. SOY-524. Escande. M. I.. Gas. N. and Stevens. B. J.. 1985. Immunolocalization of the l(H) K nucleolar protein m CHO cells. Bid. Cell. 53, !+I IO. Gocsscns, G. 1984. Nucleolar structure. Int. &I.. Cwl.. 87. 107~1SX. Goessens. G, and Lcpoint. A. 1982. Localization of Ag-NOR proteins in Ehrlich turnour cell nucleoli BIV/ Cell. 43,13Y-142. Hadjiolov, A. A. 1985. The nucleolus and ribosome hiogencsts. pp. 26X. Springer-Vcrlag. Wien. New York. Hernandez-Verdun. D.. Hubert. J.. Bourgeois. C. A. and Bouteille. M. 1980 Ultrastructural localization of Ag-NOR\ stained proteins in the nucleolus during the cell cycle and m other nuclcolar structures. Chromusoma, 79.34s-362. Herrera, A. H. and Olsen. M. 0. J. 1986. Aswciation of protein C23 with rapidly lahcllcd nucleolar RNA Aiochernisfry. 25,625%6264. Holstein. A. F. and Roosen-Runge. E. C. IYXI. Atlas of human apcrmatogcnew Grosae Vcrlag Berlin. Jordan. G. I987 At the heart of the nucleolus. Nuw-e. 329,4XY-4Y0. Lapeyre. B.. Bourbon. H. and Amalric R. lY87. Nuclcolin. the major nuclcolar protcm of growing eukaryotlc cells: An unusual protein structure revealed hy the nucleotide scqucncc. Proc. Nufl. Acud. Sri. IUSA), 84, 1472-1476. Lischwe. M. A.. Richard\. R. I. Butch. R K. and Bwch. Il. 19X1 I.ocaliration ot pho\phoprotc,n C22 to nuc~c(,lilr structures and to the nucl~olw organizer region\. trl’. (211 Rm. 136: IOI-IOY Marilley. M. and Gassend-Bonnet. Ci. lYX9. Supercoiled loop organization of gcnomic DNA : A close relationship hetwecn loop domains. expression units. and replicon organization m rDNA from Xcnopus lacvis. Erp. in the nuclcolar tihrtllar ccntct tn mciottc spcrmatocytcs and oocytc\ Pwr- .&I/. Acd. .%I. USA. SO. 5Y4&SY20 Thiry. M., Scheer. V. and Goesscns. G IYXX. Localization of DNA within Erlich turnour cell IIUCICOIIby electron microscopy. Biol. ofr/w (‘r/l. 63. 17-34.

HUMAN SPERMATOGONIA

31

Wachtler, F., Hartung, M.. Devictor, M., Wiegant, J., Stahl. A. and Schwarzacher, H. G. 1989. Ribosomal DNA is located and transcribed in the dense tibrillar component of human Sertoli cell nucleoli. Exp. Cell Res. 184, 61-71. Williams. M., Kleinschmidt, J.. Krohne, G. and Franke, W. 1982. Argyrophilic nuclear proteins of Xenopus laevis oocytes identified by gel electrophoresis. Exp. CeNRes., 137.341-351.