Chromosoma (1991) 100:162-172 C H R O M O S O M A �9 Springer-Verlag 1991

Distribution of nucleolar proteins B23 and nucleolin during mouse spermatogenesis M. Biggiogera 1, S.H. Kaufmann 1, J.H. Shaper 2, N. Gas 3, F. Amalric 3, and S. Fakan 1

1 Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland 2 The Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Centre de Recherche de Biochimie et de G~n~tique Cellulaires du C.N.R.S., F-31062 Toulouse, France

Received April 9, 1990 Accepted September 16, 1990 by M. Trendelenburg

Abstract. The intracellular distribution of nucleolar phosphoproteins B23 and nucleolin was studied during mouse spermatogenesis, a process that is characterized by a progressive reduction of nucleolar activity. Bio- chemical analyses of isolated germ cell fractions were performed in parallel with the in situ ultrastructural im- munolocalization of these two proteins by means of spe- cific antibodies and colloidal gold markers, and by silver staining. RNA blot experiments showed that mRNA for nucleolin progressively decreased during spermatogene- sis whereas mRNA for B23 increased in amount during early spermatogenic stages. Immunoblotting confirmed that both proteins were present during early spermato- genesis up to the round spermatid stage and absent from mature sperm. Immunoelectron microscopy revealed that in spermatogonia, leptotene and pachtyene sperma- tocytes, and in Golgi phase spermatids, B23 and nucleo- lin were localized in the dense fibrillar component and granular component of the nucleolus but not in the fi- brillar centers. In the dense fibrillar residue of the cap phase spermatids, labeling with anti-nucleolin but not with anti-B23 was observed. During nucleolar inactiva- tion, neither of the two polypeptides was dispersed to the nucleoplasm. Silver salts stained the fibrillar centers and dense fibrillar component but not the granular com- ponent of the nucleolus. Our results suggest that there is no direct relationship between nucleolar activity and the occurrence of B23 and nucleolin or silver staining. Moreover, we confirm that silver staining and the pres- ence of B23 or nucleolin are not directly related to each other.

Introduction

During mouse spermatogenesis, the rate of ribosomal RNA (rRNA) synthesis changes dramatically. Autora-

Offprint requests' to: S. Fakan

diographic techniques indicate a high rRNA synthetic rate in spermatogonia. This rate increases further during the first meiotic prophase to peak at the mid-pachytene stage. A progressive decrease and finally a complete ces- sation of rRNA synthesis occur as spermatogenesis con- tinues (Monesi 1965; Kierszenbaum and Tres 1975, 1978).

Striking modifications in nucleolar morphology ac- company these changes in rRNA synthetic rate. In dif- ferent types of spermatogonia the nucleolar fine struc- ture consists of normally developed nucleolonema and small fibrillar centers (FCs) but exhibits extensive vari- ability in the size, shape, and relative proportions of nucleolar components. During the first meiotic pro- phase, two or more nucleoli are found, the main or prominent one being newly formed at zygotene at the nucleolar organizing regions (NORs) of the autosomal chromosomes (Tres and Kierszenbaum 1977). The nucle- olus then migrates to the sex vesicle and at mid-pachy- tene is composed of the dense fibrillar component (DFC), the granular component (GC), a prominent FC and the round body (Solari 1969). In Golgi phase sper- matids, the nucleolus exhibits a "padlock-like" appear- ance (Krimer and Esponda 1979) and consists of an FC, a fibrogranular chord and a pale area of unknown ori- gin. In cap phase spermatids the nucleolus becomes a round dense fibrillar mass, which in later stages disap- pears completely.

The biochemical events that are responsible for these changes in nucleolar structure during spermatogenesis are incompletely understood. Two quantitatively promi- nent putative silver-binding proteins in the nucleolus have been previously identified and studied in somatic cells (Lischwe et al. 1979; for review, see Fakan and Hernandez-Verdun 1986; Hernandez-Verdun 1986). B23 [also called nucleophosmin (Chan 1989) or numatrin (Feuerstein et al. 1988)] is an Mr 37-38000 oligomeric (Fields et al. 1986; Yung and Chan 1987) phosphopro- tein that can be recovered in nuclear matrix preparations (Fields et al. 1986; Feuerstein and Mond 1987). In so-

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matic cells, levels of B23 vary with the rate of r R N A synthesis. B23 is diminished in quiescent cells such as resting lymphocytes (Feuerstein et al. 1988; Kaufmann 1989) and confluent fibroblasts (Feuerstein et al. 1988). Stimulation of these cells to proliferate leads to increased synthesis and accumulation of B23 (Feuerstein et al. 1988; Kaufmann 1989). Although the function of this polypeptide is unknown, recent evidence suggests that B23 might be an r R N A binding protein (Dumbar et al. 1989). Consistent with this view, inhibition of r R N A synthesis by serum starvation (Chan et al. 1985) or by treatment with actinomycin D or toyocamycin (Yung et al. 1985 a, b) provokes rapid redistribution of B23 out of the nucleolus and into the nucleoplasm.

Nucleolin, previously also named C23 (e.g. Lischwe et al. 1979), is also a quantitatively prominent nucleolar phosphoprotein. This Mr 100-110000 polypeptide binds preferentially in vitro to D N A sequences in the r D N A spacer regions (Olson et al. 1983) and induces chromatin decondensation by binding to histone HI (Erard et al. 1988). Previous analyses have also indicated the possible association of nucleolin with nascent pre-rRNA (Her- rera and Olson 1986). In addition, nucleolin appears to inhibit elongation of initiated r R N A transcripts until a specific proteolytic cleavage occurs (Bouche et al. 1984). As is the case with B23, levels of nucleolin increase dramatically when quiescent cells are stimulated to pro- liferate (Bouche et al. 1987).

The nucleic acid binding studies cited above suggest that B23 and nucleolin exert their functions at least in part by binding to nascent pre-rRNA and/or r D N A within the nucleolus. Other functions for these proteins remain largely unknown. Early reports (Prestayko et al. 1974) suggested that nucleolin might also be present in nonstoichiometric amounts in ribosomes. More recently, Borer et al. (1989) have demonstrated that B23 and nuc- leolin migrate from chicken nuclei to mouse nuclei in mouse/chicken heterokaryons. These results raise the in- teresting possibility that B23 and nucleolin might also shuttle between nucleus and cytoplasm during the course of carrying out their functions.

A careful ultrastructural localization of proteins B23 and nucleolin should provide further insight into their respective functions. Pre-embedding immunocytochemi- cal techniques have previously suggested that B23 and nucleolin might be present in both the D F C and the FCs (Spector et al. 1984; Ochs and Busch 1984). Using post-embedding immunocytochemistry, however, we have recently shown that B23 and nucleolin co-localize to the D F C and the GC of tissue culture cells, while FCs are completely devoid of labeling (Biggiogera et al. 1989). In the present study we extended these findings by following the fate and distribution of B23 and nucleo- lin in the nucleoli of different cells of the mouse testis. This model tissue contains somatic cells (Sertoli cells) as well as germ cells, which undergo mitosis, meiosis and transcriptional inactivation. R N A blotting, immun- oblotting, and electron microscopic immunolocalization were utilized to study B23 and nucleolin during the changes in r R N A synthesis and in nucleolar fine struc- ture that accompany spermatogenesis.

Materials and methods

Electron microscopy. A/J mice, aged 3 months, were killed by cervi- cal dislocation. The testes were immediately removed and placed into a drop of 4% paraformaldehyde in S6rensen phosphate buffer, pH 7.4. The tunica albuginea was removed and the testes cut in small pieces. The samples were then fixed in the above mixture for 2 h at 4 ~ C. After rinsing with the buffer they were placed into 0.5 M NHgC1 solution in buffer for 30 rain at room tempera- ture to block free aldehyde groups. The specimens were then rinsed in buffer, dehydrated in ethanol at progressively lower temperature and embedded in Lowicryl K4M at -35~ (Carlemalm etal. 1982). Thin sections were collected on nickel grids covered with a Formvar-carbon membrane.

The anti-B23 antibody raised in chickens (Fields et al. 1986) was affinity purified by adsorption to nitrocellulose-immobilized B23 and release by sodium thiocyanate as described by Kaufmann (I 989). The anti-nucleolin polyclonal antibody was raised in rabbit using the Mr 100000 protein as immunogen (Lapeyre et al. 1985).

The grids with ultrathin sections were placed on drops of PBS (phosphate buffered saline, pH 7.4) containing normal goat serum (NGS) diluted 1:100 for 3 rain at room temperature. They were then placed on 40 gl droplets of PBS containing 0.1% bovine serum albumin (BSA, fraction V, Merck), 0.05% Tween 20 (Sigma) and one of the two antibodies at a 1 : 50 dilution, for 17 h at 4 ~ C. The specimens were then rinsed with PBS containing 0.05% Tween 20, and then with PBS. For detection of protein B23, it was necessary to incubate the sections with a secondary rabbit anti-chicken IgG antibody (affinity purified, EY Labs) diluted t : 50 with PBS containing Tween and BSA for 30 min at room tempera- ture.

After rinsing with PBS, all the grids were placed on droplets of NGS as above and labeled for 30 rain at room temperature with a 1:20 dilution (in PBS) of affinity purified goat anti-rabbit IgG coupled with 15 nm colloidal gold (Janssen Life Sciences). All the grids were then sequentially rinsed with PBS and distilled water. After air drying, they were stained with uranyl acetate and lead citrate.

As controls, some grids were floated on the incubation mixture from which the primary antibody was excluded, and then processed as above. In addition, in the case of the anti-B23 antibody, some grids were also incubated in the absence of both the primary anti- body and rabbit anti-chicken IgG bridge antibody.

For silver staining sections were collected on gold grids and silver stained by the one-step technique (Moreno et al. 1985). They were then briefly contrasted with uranyl acetate.

For specific detection of DNA, ultrathin sections were collected with plastic rings, hydrolyzed for 20 min at room temperature with 5 N HC1, rinsed with water and stained with an osmium ammine complex (0.1% in water) for 90 min at room temperature (Cogliati and Gautier 1973).

Staining of both DNA and RNA was done by floating the grids first on a solution of ethidium bromide in water (50 ~tg/ml) for 30 min at room temperature and then on 1% neutralized phosp- hotungstic acid (PTA) for 30 rain (EB-PTA; Biggiogera and Flach Biggiogera 1989).

All the grids were observed with a Philips EM 300 or Philips CMI0 electron microscope operating at 80 kV and equipped with a 3040 ~tm objective aperture.

Immunoblotting. Mouse liver nuclei were isolated and solubilized for immunoblotting using techniques previously applied to rat liver nuclei (Kaufmann 1989).

Populations of mouse pachytene spermatocytes and round spermatids prepared by elutriation as previously described (Shaper et al. 1990) were kindly provided by Dr. William Wright (Johns Hopkins University School of Public Health). Mouse spermatozoa were collected from the cauda epididymidis. Germ cells were washed once in protein-free buffer (Toyoda and Chang 1974) and solubilized by sonication in 6 M guanidine hydrochloride contain- ing 250 mM Tris-HC1, pH 8.5, 10 mM EDTA, 1% (v/v) 2-mercap-

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toethanol and lmM phenylmethylsulfonylfluoride (freshly added from a 100 mM stock in anhydrous isopropanol). After alkylation with iodoacetamide, samples were dialyzed sequentially into 4 M urea and 0.1% (w/v) SDS. Samples were lyophilized and resus- pended in small volumes of electrophoresis sample buffer contain- ing 4 M urea, 2% (w/v) SDS, 62.5 mM Tris, pH 6.8 at 21 ~ C, and 1 mM EDTA. Samples derived from a fixed number of cells (1 x 10 6) were applied to various lanes of the gel. To provide a standard curve for comparison, additional samples containing various dilu- tions of mouse liver nuclei (ranging from 5 x 104 to 2 x 106) were applied to additional wells of the same gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent electrophoretic transfer to nitrocellulose were performed as previously described (Kaufmann et al. 1987). Chicken antibodies against B23 (Fields et al. 1986), lamins A and C or lamin B (Kaufmann 1989) and rabbit antibodies against nucleolin (Bugler et al. 1982) were utilized for immunoblotting (Kaufmann et al. 1987).

RNA blot analysis. RNA was prepared from elutriated germ cells or somatic cells by the guanidinium thiocyanate method and sub- jected to electrophoresis on formaldehyde-containing agarose gels as previously described (Shaper et al. 1990). Each lane contained 10 ~g total RNA. Duplicate blots containing the same RNA prepa- rations were utilized in this study. Plasmid U9-SP6 containing the 1300 bp coding sequence for B23 (kindly provided by Dr. P. Chan, Baylor College of Medicine, Houston, Tex.) and plasmid BL16.1 (Lapeyre et al. 1985) containing an 850 bp fragment from the cod- ing sequence of nucleolin were digested with EcoRI (Bethesda Re- search Laboratories, Bethesda, Md.). Inserts were purified by elec- trophoresis on agarose gels, recovered by electroelution, and la- beled by the random primer method (Feinberg and Vogelstein 1983). RNA blotting was performed as recently described (Shaper et al. 1990). Autoradiography was performed using preflashed Ko- dak XRP-5 (B23) or XAR-5 (nucleolin) film and appropriate inten- sifying screens.

Results

RNA blot analysis of B23 and nueleolin mRNA in germ eell populations

R N A blot analysis (Fig. 1) was utilized to analyze R N A f rom purified popula t ions o f germ cells for the presence o f nucleolin and B23 m R N A s . The nucleolin c D N A hy- bridized to a single 2.6 kb m R N A species in germ cells (Fig. 1 A, lanes 1-4) and somatic cells (Fig. 1 B, lane 5). The a m o u n t o f nucleolin m R N A was the greatest in spe rmatogon ia and progressively decreased t h rough the r o u n d spermat id stage (Fig. 1 A, lanes 1-4, respectively). In contrast , c D N A for B23 hybridized with a single 1.4 kb m R N A (Fig. 1 B) tha t progressively increased dur ing early spermatogenesis (lanes 1-4, respectively). These results suggest tha t nucleolin and B23 might be differentially regulated dur ing the early stages o f sper- matogenesis .

Immunoblot analysis of B23 and nucleolin in germ cell populations

The presence and relative levels o f nucleolin and B23 in enriched popula t ions o f specific germ cell stages were examined by immunob lo t t i ng (Fig. 2). Nucleol in/C23 (Fig. 2B) was detectable as an M r 100-110000 double t in mouse liver (lane 5) and mouse pachytene spermato-

Fig. 1 A, B. Detection of B23 and C23/nucleolin mRNA during spermatogenesis. Samples containing mouse spermatogonia (lane 1), early spermatocytes (33% type B spermatogonia, 66% preleptotene spermatocytes) (lane2), pachytene spermatocytes (lane 3), round spermatids (lane 4) or mouse C127 cells (lane 5) were lysed in guanidine thiocyanate and subjected to RNA blot analysis as described in Materials and methods. Two identical blots using the same RNA samples were utilized in this figure. Rehybridi- zation with the probe for glyceraldehyde-3-phosphate dehydroge- nase confirmed that the levels of hybridizing RNA were similar in all lanes (data not shown). Nonadjacent lanes from a single autoradiograph have been juxtaposed to compose each panel. The cDNA for C23/nucleolin (A) hybridizes to a 2.6 kb mRNA species. This mRNA appears relatively more abundant in spermatogonia (lane I) and progressively diminishes during spermatogenesis (lanes 2-4). The cDNA for B23 (B) hybridizes to a single 1.4 kb mRNA species. The signal for this species is low in spermatogonia (lane 1) and increases during early spermatogenesis (lanes 2-4)

cytes (lane 1). The origin o f the two species with slightly different migra t ions on unidimensional S D S - P A G E is at present unknown . Nucleol in is k n o w n to be sensitive to proteolysis in vitro (Bugler et al. 1982). Alternatively, nucleolin is also a phosphopro te in . Differences in the phosphory la t ion state o f some proteins have been shown to result in altered mobil i ty on S D S - P A G E (Smith and Fisher 1989). Wha tever the explanat ion, bo th species were markedly diminished by the time germ cells reached the r o u n d spermatid stage (cf. Fig. 2 B, lanes 1 and 2). Compar i son with a series o f dilutions o f mouse liver

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nuclei (not shown) revealed that the level of nucleolin had diminished at least fourfold during the course of meiosis and that it was undetectable in mouse epididy- real sperm (Fig. 2 B, lanes 3, 4).

B23 (Fig. 2C) was also detected as a doublet in mouse liver (lane 5) and pachytene spermatocytes (lane 1). This doublet for B23 (also observed in Chan et al. 1985; Yung and Chan 1987) reflects two different- sized but closely related polypeptides (Chan et al. 1986). In contrast to nucleolin, the signal for B23 does not diminish by the round spermatid stage. During further germ cell maturation, however, B23 diminishes marked- ly. B23 was barely detectable in mature epididymal sperm (Fig. 2 C, lane 4).

Electron microscopic immunolocalization o rB23 and nucleolin during spermatogenesis

After confirming the presence of nucleolin and B23 in germ cells (Fig. 2), we next determined the ultrastructur- al localization of these polypeptides during the various changes in nucleolar morphology that accompany sper- matogenesis. Sertoli cells present on the same grids served as a somatic cell control. The general pattern of immunolabeling with the anti-B23 and anti-nucleolin antibodies clearly demonstrates that most label is con- centrated over nucleoli, regardless of the differentiation stage examined. While a low level of nucleoplasmic label- ing can be observed after incubation of sections with the anti-nucleolin antibody, the nucleoplasmic labeling with anti-B23 as well as the cytoplasmic signal found after incubation with both the above antibodies, remain very low and are comparable with the background de- tected on the sections outside the cells. In all the control sections incubated in the absence of the primary anti- bodies, the number of gold grains present as background was negligible.

In the nucleoli of Sertoli cells, the pattern of immun- olabeling of the two proteins as well as of the silver

Fig. 2 A-E. Detection of B23 and C23/nucleolin in maturing mouse germ cells by immunoblotting. Samples containing mouse pachy- tene spermatocytes (lane 1), round spermatids (lane 2) mouse epidi- dymal spermatozoa (lanes 3, 4), or mouse liver nuclei (lane 5) were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie blue (A) or transfer to nitro- cellulose followed by immunoblotting (B-E). Antibodies against C23/nucleolin (B), B23 (C), lamins A and C (D) or lamin B (E) were utilized in these studies. Lanes 1-3 and 5 were loaded with samples derived from 1 x 1 0 6 cells or nuclei; lane 4 was loaded

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with protein derived from 2 x 104 spermatozoa. Numbers at left refer to molecular weights of standard proteins in kDa. H indicates core histones (apparent molecular weight 12-14 kDa). C23/nucleo- lin (B) and B23 (C) were readily detected in mouse pachytene sper- matocytes (lane 1) and round spermatids (lane 2). C23/nucleolin was undetectable in mature spermatozoa (B, lane 4) ; and the level of B23 (C, lane 4) was markedly diminished in spermatozoa (<2% the amount detected in an equal number of mouse liver nuclei; see Materials and methods for quantitation). To rule out the possi- bility of contamination of germ cells by somatic cells, blots were probed with antibodies to lamins A and C, polypeptides that have been shown to be absent from rodent germ cells (Moss et al. 1987; Kaufmann 1989). Lamins A and C were undetectable in the pachy- tene spermatocytes (D, lane 1) and round spermatids (D, lane 2). This result is consistent with the absence of contaminating somatic cells in these germ cell populations. In contrast, lamin B was readily detected in these maturing germ cells (E, lanes 1 and 2) but marked- ly diminished in mouse spermatozoa (< 2% the amount detected in an equal number of mouse liver nuclei)

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staining was the same as that described previously for HeLa cell nucleoli (Biggiogera et al. 1989). The two anti- bodies were localized over the nucleolar DFC and GC. The FCs were devoid of any labeling. Silver staining of Sertoli cell nucleoli showed the reaction product to be present on the DFC and the FCs, while the GC re- mained unstained (results not shown).

In spermatogonia, the nucleolus is often present in the form of threads composed of the GC and the DFC and characterized by few and small FCs. One small FC is recognizable in Fig. 3. In these cells the labeling pat- tern was found to be the same for the two antibodies. Both the DFC and the GC were labeled, whereas no labeling was found in the FCs. The silver staining pattern was similar to that described for Sertoli cells (Fig. 4).

A newly formed nucleolus becomes recognizable in the primary spermatocyte during pachytene, the longest of the meiotic phases. This phase is characterized by the presence of synaptonemal complexes (SCs) connect- ing the sister chromatids and linking them to the nuclear envelope. In Lowicryl-embedded tissue, condensed chro- matin structures like chromosomes display light gray contrast, while the SCs appear strongly stained. Pachy- tene can be subdivided into three distinct subphases: early, mid and late. In early pachytene, the newly formed nucleolus is associated with the NORs of the autosomes (Fig. 5). In mid-pachytene, the nucleolus migrates to- ward the sex vesicle formed by the X and Y chromo- somes near the nuclear membrane (Fig. 6). At this stage the DFC and the GC can be observed surrounding the sex vesicle. Both the DFC and the GC are labeled by the two antibodies. Finally, in late pachytene spermato- cytes, a complete nucleolus is present as a structure in which the DFC and the GC surround the sex vesicle and where the central part consists of the FC and of a round gray homogeneous structure of yet unknown function, termed the round body (Solari 1969). The pat- tern of immunolabeling (Fig. 7 a) and of silver staining (Fig. 7b) of the nucleolus at this stage is not modified compared with the previous stages. The DFC and GC are labeled with anti-B23 and anti-nucleolin antibodies (Fig. 7a). In comparison with the other pachytene phases, the labeling of the DFC and GC with both anti- bodies is markedly increased. An FC (indicated by an arrow and not labeled by the antibody) is surrounded by the DFC and is located in the vicinity of the round body. The round body was never found labeled either by the anti-B23 or anti-nucleolin antibody. In addition, the round body does not stain with silver (Fig. 7 b), with the osmium ammine stain for DNA (Fig. 8), or with the EB-PTA technique for DNA and RNA (Fig. 9).

In diplotene spermatocytes the immunolabeling and cytochemical properties of the nucleolus do not change.

After the first meiotic division, the spermatocytes II divide almost immediately. Thereafter, the fate of the nucleolus can be followed for some time in spermatids. In Golgi phase spermatids (steps 1-3 according to Oak- berg 1956) the nucleolus is present in a form of one to three structural entities called padlock-like nucleoli (Krimer and Esponda 1979). In such a nucleolus, a fibro- granular chord surrounds a fibrillar sphere containing

Fig. 3. The nucleolus of a spermatogonium after labeling with the anti-B23 antibody. Note that the gold grains are present over the granular component (GC; long arrow) as well as over the dense fibrillar component (DFC; arrowhead) but not on the fibrillar center (FC; small arrow). Bar represents 1 gm Fig. 4. After silver staining, the nucleolus of a spermatogonium is stained over the DFC (D) and the FC (arrowhead), the GC (arrow) being devoid of any staining. Bar represents 1 Ixm

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Fig. 5. Early pachytene spermatocyte, labeled for protein nucleolin. The GC (G) and the DFC (arrow), constituting most of the nucleo- lus, are both labeled. Bar represents 1 pm

Fig. 6. Mid-pachytene spermatocyte. Anti-B23 antibody labeling. A labeled nucleolus has reached the sex vesicle (asterisk). Bar repre- sents 1 pm

Fig. 7. a Late pachytene spermatocyte labeled for protein nucleolin. The labeling is present over the DFC (arrowhead) and the GC (G). Also in this case, the FC (arrow) and the round body (R) are not labeled, b A nucleolus at the same cell stage after silver staining. Note the heavily stained FC (arrow) and the unstained round body (R). Bar represents 0.5 ~tm

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the FC and a pale, grayish structure similar to the round body. The fibrogranular chord is weakly labeled by the two antibodies, while both the internal structures are virtually devoid of labeling (Fig. 10a, b). In contrast, staining with silver salts results in heavy deposits of silver over the FC and a fine deposit of silver over the chord while the pale area remains unstained (Fig. 10 c; see also Mirre and Knibiehler 1985; Fakan and Hernandez-Ver- dun 1986).

In cap phase spermatids the nucleolus is completely condensed to form a dense, homogeneous mass. Label- ing was observed with the anti-nucleolin antibody (Fig. l ib) , but not with the anti-B23 antibody (Fig. l la). This dense nucleolar mass is, however, strongly and homogeneously stained with silver (Fig. 11 c).

Later in spermiogenesis, the nucleolus disappears as a recognizable structure. Immunolabeling with the two antibodies and silver staining are no longer detectable in the maturing spermatid nucleus.

Fig. 8. After specific staining for DNA, the pachytene chromo- somes and intranucleolar chromatin are stained. Both the nascent nucleolus (arrow) and the round body (R) only display their inher- ent contrast. Bar represents 0.5 gm

Fig. 9. A round body (R) is shown after ethidium bromide-phosp- hotungstic acid staining for both DNA and RNA. It is not stained but the precipitates are present over the nucleolus (arrow) as well as over the chromatin. Bar represents 0.5 [xm

Discussion

Previous studies on somatic cells have revealed that the levels of B23 and nucleolin, two of the putative silver- binding proteins of the nucleolus, are low in quiescent cells and increase dramatically after stimulation to pro- liferate (Feuerstein et al. 1988; Bouche et al. 1987; La- peyre et al. 1987). Since rRNA synthesis also increases after mitogenic stimulation (Kelly et al. 1983), these ob- servations suggest that levels of B23 and nucleolin might be functionally linked to rRNA synthesis. If this were the case, then one might predict that levels of B23 and nucleolin would diminish as the rate of rRNA synthesis decreased. Experiments in neoplastic tissue culture cells, however, have revealed that inhibition of rRNA synthe- sis leads to a redistribution of B23 from the nucleolus to the nucleoplasm without much change in the total nuclear level of the protein (Chan etal. 1985; Yung et al. 1985 a, b). In contrast, nucleolin remains in the nucleolus after inhibition of rRNA synthesis (Chan et al. 1985; Yung et al. 1985a, b; Escande-G6raud et al. 1985).

The fate of B23 and nucleolin has not been previous- ly described during the course of nucleolar inactivation that occurs as a normal consequence of cellular differen- tiation in vivo. In the present study we utilized RNA blotting, immunoblotting and ultrastructural localiza- tion to follow the fate of B23 and nucleolin during the process of nucleolar inactivation that accompanies mouse spermatogenesis. This model system was chosen because the sequential changes in the rate of rRNA syn- thesis (with its peak in mid-pachytene and subsequent progressive decline) have been described in detail (Mone- si 1965; Kierszenbaum and Tres 1975, 1978) and because reproducible changes in nucleolar morphology have been observed to accompany the process of nucleolar inactivation (Kierszenbaum and Tres 1975). We attempt- ed to correlate these changes in rRNA synthetic rate and nucleolar morphology with changes in the levels and ultrastructural distribution of B23 and nucleolin.

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Fig. 10. a In the padlock-type nucleolus of spermatids in Golgi phase, the fibrogranular chord (arrow) is labeled with the anti-B23 antibody while the pale area (P) is not. b After labeling for nucleo- lin, the pattern is similar to that in a, with the labeling present only on the fibrillar component at the periphery of the nucleolus (arrowhead). The FC (arrow) and the pale area (P) are devoid of labeling, e A padlock-like nucleolus after silver staining. Note the heavy staining over the fibrillar component and the FC (arrow) and its absence on the pale area (P). The fibrogranular chord (ar- rowhead) is weakly stained. Bars represent 0.5 gm

Fig. 11. a Cap phase nucleolus. Labeling for protein B23. The con- densed nucleolus is almost unlabeled, b The same stage as in a but labeling with the anti-nucleolin antibody. The labeling over the nucleolus is significant, e Cap phase nucleolus after silver stain- ing. The condensed nucleolus is heavily stained. Bars represent 0.5 gm

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We observed that levels of nucleolin mRNA are high- est early in spermatogenesis and progressively decline prior to pachytene (Fig. I A). In contrast, levels of mRNA for B23 progressively rise as spermatogonia ma- ture to round spermatids (Fig. 1 B). These results might indicate either differential stability of the two mRNAs or differential expression of the respective genes.

Levels of B23 and nucleolin determined by immun- oblotting in pachytene spermatocytes are similar to lev- els observed in metabolically active somatic cells (cf. Fig. 2, lanes 1 and 5). Both proteins are markedly dimin- ished in amount in mature sperm (Fig. 2, lanes 3 and 4).

Ultrastructural immunocytochemistry permitted the study of the distribution of B23 and nucleolin during the process of nucleolar inactivation and involution. In pachytene spermatocytes, B23 and nucleolin were ob- served to co-localize in the DFC and GC (Figs. 5-7a). Both proteins were absent from the FCs. This distribu- tion of B23 and nucleolin is similar to that described in dividing CHO cells (Escande et al. 1985), HeLa cells (Biggiogera et al. 1989), and Sertoli cells (this report). As rRNA synthesis diminished, B23 and nucleolin re- mained localized to the DFC and GC in Golgi phase spermatids (Fig. 10a, b). We did not find redistribution of B23 out of the nucleolus and into the nucleoplasm. In this regard, our results observed during the process of the nucleolar inactivation in vivo are in sharp contrast to results obtained after serum starvation or drug treat- ment of neoplastic cells in vitro (Chan et al. 1985; Yung et al. 1985 a, b). As nucleolar inactivation progressed fur- ther, the nucleolar remnant in cap phase spermatids con- tinued to be labeled with anti-nucleolin antibody (Fig. 11 b) but the signal for B23 disappeared completely (Fig. 11 a). Finally, both proteins were absent when the nucleolar remnant could no longer be detected ultra- structurally.

It is interesting to assess whether the levels of B23 and/or nucleolin are directly related to the level of nuc- leolar activity during mouse spermatogenesis. Nucleolar activity is highest during pachtyene and markedly dimin- ished by the round spermatid stage (Monesi 5965; Kiers- zenbaum and Tres 1975, 5978). Levels of B23 do not change between pachytene and the round spermatid stage (Fig. 2C) but levels of nucleolin diminish about fourfold during this time period (Fig. 2B). With further maturation to cap phase spermatids, rRNA synthesis ceases completely (Monesi 1965; Kierszenbaum and Tres 5975, 1978), but the labeling for nucleolin/C23 persists (Fig. 11 b). Neither the level of B23 nor the level of nuc- leolin/C23 completely parallels the level of nucleolar ac- tivity. However, both proteins do eventually disappear when the nucleolus as a morphological entity can no longer be discerned.

The results obtained with specific antibodies against B23 and nucleolin can be compared with previous obser- vations on the silver staining of nucleoli during sperma- togenesis. In spermatogonia, spermatocytes, and Golgi phase spermatids, silver staining gives a positive reaction in the FC and DFC but not in the GC of the nucleolus (Figs. 4, 7b, 10c, 11c). These data are in substantial

agreement with earlier findings (Knibiehler and Mirre 1983; Mirre and Knibiehler 1985; Czaker 1987). All of these investigations indicate that nucleolar components can be stained with silver from the meiotic prophase up to the elongating spermatids. In fact, mid-pachytene spermatocytes (in which rRNA synthesis has reached a peak) and Golgi or cap phase spermatids (in which nucleolar activity has been reduced or arrested; see Fak- an and Hernandez-Verdun 1986) stained similarly with silver salts (cf. Figs. 7c and 11c). These observations lead to the conclusion that silver staining also bears no direct relationship with the activity of the nucleolus.

Several points require emphasis concerning the struc- ture of the nucleolus in Golgi phase spermatids. The observations of Mitre and Knibiehler (1985; see also Fakan and Hernandez-Verdun 1986) show that in these cells the padlock-like nucleoli are composed of two adja- cent spherical structures. One structure is strongly stained with silver and consists of an FC surrounded by a dense fibrillar layer. The second structure is consti- tuted by a moderately silver-stained fibrogranular chord surrounding a finely fibrillar pale area devoid of silver. Our findings after silver staining and immunolabeling are in agreement with these observations (see Fig. 10a- c). However, they disagree with the conclusions of other reports (Krimer and Esponda 1979; Czaker 1987). The conclusions of these reports can lead to a possible misin- terpretation of the silver staining experiments as a result of an apparent confusion between the FC and the pale area in the padlock-type nucleoli. In addition, we would like to emphasize the similarity (previously noted by Mitre and Knibiehler 1985) between the round body described by Solari (1969) and the round clear area pres- ent inside the padlock-type nucleolus of Golgi phase spermatids. These structures are neither labeled with the two antibodies nor stained with silver or the osmium ammine complex specific for DNA (Fig. 8). The nature and possible functions of these two structural compo- nents still remain to be elucidated.

Finally, our results show that staining with silver salts does not parallel the labeling with either the B23 or the nucleolin antibody. First of all, the FCs of Sertoli cells and early germ cells stain with silver but not with antibodies to B23 or nucleolin. This observation suggests the presence in the FCs of another nucleolar compo- nent(s) capable of reducing silver salts (Biggiogera et al. 1989). Conversely, the GC labels with antibodies against both B23 and nucleolin but is devoid of silver reaction product. If B23 and nucleolin were indeed among the major silver-staining nucleolar components, one would have to postulate that post-transcriptional modification of these proteins (e.g., dephosphorylation or sulfhydryl oxidation) occurs in the GC and alters the ability of both these proteins to reduce silver salts. Alternatively, the possibility that B23 and nucleolin are not the pro- teins that are responsible for silver staining in situ should be considered. Further studies are required to distinguish between these two alternatives.

Acknowledgements. We gratefully acknowledge the kind gift of pur- ified populations of mouse germ cells from Dr. W. Wright, the

171

gift of plasmid U9-SP6 from Dr. P. Chan, the patient assistance of Dr. Nancy Shaper in performing the RNA blot analysis, the technical assistance of Ms. Sandra Kiesewetter and the photo- graphic work carried out by Ms. Corinne Cottier. These studies were supported by grants from the Swiss National Science Founda- tion (3.355.1.86), the N.I.H. (CA06973, GM10518), the American Cancer Society and the Andrew Mellon Foundation. M.B. is a recipient of a fellowship from the Fondation pour des Bourses d'Etudes Italo-Suisse, Lausanne, Switzerland.

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