Development 110, 1263-1270 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

1263

Nucleoiar distribution of proteins B23 and nucleoiin in mouse

preimplantation embryos as visualized by immunoeiectron microscopy

M. BIGGIOGERA1, K. BURKI2, S. H. KAUFMANN3, J. H. SHAPER3, N. GAS4, F. AMALRIC4 and

S. FAKAN1'*lCentre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005 Lausanne, Switzerland2Pre-CIinical Research, Sandoz Pharma Ltd., CH-4002 Basel, Switzerland3The Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA4Centre de Recherche de Biochimie et de Cenetique Cellulaires du C.N.R.S., 31062 Toulouse, France

* Author for correspondence

Summary

The ultrastructural distribution of proteins B23 andnucleoiin in the nucleolus of mouse embryos from thezygote to the early blastocyst has been analyzed bymeans of specific antibodies and immunocytochemistryusing colloidal gold complexes as markers. In parallel,silver staining of nucleoli was carried out on ultrathinsections. Our results show that the compact prenucleolarbodies at 1- and 2-cell stage as well as the compactresidual fibrillar masses observed up to the morulastage, are labelled with the two antibodies. Thesemasses, however, are not stained with silver up to the4-cell stage. In well-developed nucleoli, the two anti-bodies co-localize in the dense fibrillar component (DFC)

and the granular component (GC) while fibrillar centers(FCs) are devoid of label. On the contrary, silverstaining occurs in the FCs and DFC but not in the GC.Our observations suggest that there is no directrelationship between the occurrence of silver stainingand the distribution of protein B23 or nucleoiin.Moreover, neither the localization of the two aboveproteins nor silver staining are unequivocally related tothe nucleoiar activity.

Key words: nucleolus, mouse early embryos,immunoeiectron microscopy, silver staining.

Introduction

Preimplantation mammalian embryos represent anexcellent model of nucleoiar reactivation. In the mouse,the nucleolus of the zygote is essentially a round, denseand compact mass also called prenucleolar body. Nointernal structural compartmentation can be detected atthis time. A small fibrillar area, supposed to representthe fibrillar center in these nucleoli, can occasionally beobserved on the periphery of the prenucleolar bodies(Takeuchi and Takeuchi, 1986). The beginning of anewnucleoiar activity accompanied by changes in themorphology of the prenucleolar body varies accordingto the species. In the mouse, it essentially takes placeduring the transition from 2 to 4 blastomeres (Wood-land and Graham, 1969; Geuskens and Alexandre,1984) while, in human embryos, the first signs ofnucleoiar activity appear after the 4-cell stage (Tesariketal. 1986, 1987; Tesarik and Kopecny, 1989a,fc) and, inthe cow, in the 8-cell stage (Kopecny et al. 1989).

The initiation of the nucleoiar activity in the embryohas been studied mainly by autoradiography afterincorporation of labelled RNA precursors (Mintz, 1964;

Bernstein and Mukherjee, 1972; Geuskens and Alex-andre, 1984; Camous et al. 1986). A technique widelyused to visualize the nucleoiar organizer regions or theirinterphase counterpart is silver staining. In embryonucleoli, stained fibrillar centers (FCs) have beenshown as early as the zygote stage in the mouse(Takeuchi and Takeuchi, 1986). It must be noted,however, that the reasons for the silver staining of thenucleolus are still poorly understood. Clusters of thiolgroups in proteins have been suggested to be at thebasis of the reaction (De Capoa et al. 1982). Twonucleoiar acidic phosphoproteins, B23 and nucleoiin,exhibit such properties (Satoh and Busch, 1981) andhave been suggested as the major silver-stainingproteins (Lischwe et al. 1979). However, neither theirrole in the nucleolus nor their localization with regardsto different nucleoiar components have been clearlydemonstrated. Protein B23 has been recovered innuclear matrix fraction preparations (Fields et al. 1986)and its abundance seems to be related to cellproliferation (Feuerstein and Mond, 1987). Nucleoiin(also called C23) has been shown to bind preferentiallyin vitro to spacer regions of rDNA (Olson et al. 1983),

1264 M. Biggiogera and others

while other reports suggested its association withnascent pre-rRNA or its processing products (Herreraand Olson, 1986; Bugler et al. 1987). Possible shuttlingof proteins B23 and C23 between the nucleus and thecytoplasm and their involvement in the nucleocytoplas-mic transport of ribosomal components have recentlybeen considered (Borer et al. 1989).

Previously (Biggiogera et al. 1989), we have shownthat the fine structural distribution of these proteins asvisualized by specific antibodies follows only partiallythe silver-staining pattern in exponentially growingHeLa cell nucleoli. Similar results were obtained onmouse testis cells, a tissue model in which nucleoliundergo, in the course of spermiogenesis, physiologicalinactivation (Biggiogera et al. 1990).

The aim of the present work is to follow theultrastructural distribution of the two phosphoproteinsin the prenucleolar bodies and in the nucleoli of mousepreimplantation embryos by means of specific anti-

bodies. By correlating the immunocytochemical datawith those obtained using the silver-staining techniquesin nucleoli undergoing physiological reactivation, weattempt to understand better the relationship betweenthese proteins and the silver reaction as well as theirrole in nucleolar functions.

Materials and methods

Female mice (strain B6D2F1), 4-6 weeks old, were injectedwith pregnant mare serum (Folligon, Intervet, 5i.u. ml"1 inPBS) and 46 h later with human chorionic gonadotropin(Chorulon, Intervet, 5 i.u. ml"1 in PBS) and mated with malesof the same strain. The embryos were recovered by flushingthe oviducts or uterus with Hank's solution 12 h (zygote), 24 h(2-cell), 48 h (4-8 cell), or 72 h (morula-early blastocyst) afterthe presumed time of fertilization (12 h post-HCG). Theembryos were then fixed with 4% paraformaldehyde inSorensen phosphate buffer (pH7.4) at 4°C for 2h, rinsed

/

\

- «p

\ • • • * •

'•i'

Fig. 1. Distribution of silverstaining at the zygote stage.Limited peripheral areas(arrows) are stained by silversalts, while the prenucleolarbody is devoid of silver,x 50 000.Fig. 2. Distribution of anti-nucleolin labelling at thezygote stage. The gold grainsare scattered over the denseprenucleolar body. Theperipheral areas correspondingto silver-stained material(arrows) are devoid of grains,x 50 000.

B23 and nucleolin in early mouse embryos 1265

several times with the same buffer and pre-embedded in lowviscosity agarose. Some specimens were then placed into 0.5 MNH4CI solution in buffer for 30min at room temperature toblock free aldehyde groups. Some embryos, used for silverstaining, were fixed with 1% glutaraldehyde, lh, 4°C. Allsamples were dehydrated in ethanol at progressively lowertemperature and embedded in Lowicryl K.4M (Carlemalm etal. 1982).

ImmunocytochemistryThe anti-B23 antibody was raised in chicken by using rat liverB23 purified protein (Fields et al. 1986). The antibody wasaffinity purified by absorption to the nitrocellulose-immobi-lized 38xlO3Mr polypeptide and release by thiocyanate, aspreviously described (Fields et al. 1986; Kaufmann, 1989).The anti-nucleolin polyclonal antibody was raised in rabbit byusing purified nucleolin from a Chinese hamster ovary cell line(CHO) and the IgG fraction was purified as previouslydescribed (Lapeyre et al. 1985). These antibodies havepreviously been demonstrated by means of Western blottingor immunoelectron microscopy to cross-react with mouse B23and nucleolin (Biggiogera et al. 1990).

Thin sections, collected on nickel grids covered with aFormvar-carbon membrane, were placed on drops of PBS(pH7.4) containing normal goat serum (NGS) diluted 1:100,for 3min at room temperature. They were then placed on40jul droplets of PBS containing 0.1 % bovine serum albumin(BSA, fraction V, Merck), 0.05 % Tween 20 (Sigma) and oneof the two antibodies at a 1:50 dilution, for 17 h at 4°C. Thespecimens were then rinsed with PBS containing 0.05 %Tween 20, and then with PBS.

For detection of protein B23, it was necessary to incubatethe sections with a secondary, rabbit anti-chicken IgGantibody (affinity purified, Nordic) diluted 1:50 with PBS

containing Tween and BSA as above, for 30min at roomtemperature. After rinsing with PBS, all the grids were placedon droplets of NGS as above and finally labelled with affinity-purified goat anti-rabbit IgG coupled with 15 nm colloidalgold (Janssen Life Sciences) diluted 1:20 with PBS, for30minat room temperature.

As controls, some grids were floated on the incubationmixture from which the primary antibody was excluded, andthen processed as above. In addition, in the case of the anti-B23 antibody, some grids were also incubated in the absenceof the primary antibody and of the rabbit anti-chicken IgG.All the grids were finally rinsed with PBS and distilled water.They were then stained with uranyl acetate and lead citrate.

Silver stainingSome of the thin sections from formaldehyde- or glutaralde-hyde-fixed samples were collected on gold grids and silverstained with the one-step technique (Moreno et al. 1985).They were then briefly contrasted with uranyl acetate.

All specimens were observed with a Philips EM 300 orPhilips CM10 electron microscopes operating at 80 kV andequipped with a 30-40 j.im objective aperture.

Results

In the zygote, the nucleolus or prenucleolar body is avery dense homogeneous and contrasted structure.While the prenucleolar bodies appear unstained aftersilver treatment, strong silver reaction occurs on smallareas occasionally observed at their periphery (Fig. 1).These areas are, however, difficult to find on sections offormaldehyde-fixed, Lowicryl-embedded material, astheir internal structure appears highly dispersed.

Fig. 3. Labelling with the anti-B23 antibody at the 2-cell stage.Few gold grains are present overthe dense nucleolar mass,x 28 000.Fig. 4. Labelling with the anti-nucleolin antibody at the 2-cellstage. The labelling appearshigher when compared withFig. 3. X28000.

1266 M. Biggiogera and others

Nevertheless, we have been able to observe them inspecimens incubated with the anti-nucleolin antibodyand revealed the absence of labelling at these particularareas. The prenucleolar masses were labelled with theanti-nucleolin (Fig. 2) as well as the anti-B23 anti-bodies. In the two cases, the gold grains are regularlyscattered on the compact nucleolus. The nucleoplasm isnot significantly labelled (Fig. 2).

At the 2-cell stage, the compact fibrillar mass remainsunstained after the reaction with silver. After labellingwith the anti-B23 antibody, the number of gold grainsobserved over the dense mass is considerably lowerthan in the zygote (Fig. 3). However, after labellingwith the anti-nucleolin antibody, the labelling densityremains similar to that of the zygote (Fig. 4).

Beginning with the 4- to 8-cell stage, the nucleolusslowly decondenses giving rise to a fibrogranularnetwork emerging from the core structure which

remains still condensed. The dense fibrillar component(DFC) and sometimes the fibrillar centers (FCs) arerecognizable. After immunolabelling with the twoantibodies, the gold grains are present on the stillcondensed area as well as on the nucleolonemalnetwork. The labelling pattern and density are compar-able for the two probes. In Fig. 5, the labelling ispresent on the DFC and on the granular component(GC). Fibrillar centers are rare and they are neverlabelled. Silver staining of nucleoli at this stage resultsin a finely granular deposit of silver distributed on thedense core; on the periphery, the DFC and FC areheavily stained whereas the GC remains free of silverdeposits (Fig. 6).

The morula nucleoli differ morphologically fromprevious stages. The condensed core is fragmented intoseveral small areas connected by the network of theDFC and GC. The two proteins co-localize on these two

\f *

v*.

Fig. 5. 4- to 8-cell stage afterlabelling with the anti-B23antibody. The nucleolus startsdecondensing on its peripheryand the newly formednucleolonema is labelled. Thegold grains are still present onthe central, dense mass,x 28 000.Fig. 6. Distribution of silverstaining at 4- to 8-cell stage.The dense mass is coveredwith fine silver precipitates. Inthe nucleolonema, the DFC(arrowheads) and the FC(arrow) are heavily stained,x 29 000.

B23 and nucleolin in early mouse embryos 1267

*i«v v* % w 'iff*

7 v -•>

* •8

latter constituents. The residual condensed core massesare virtually unlabelled (Fig. 7). The silver-stainingpattern is similar to that of the 4-cell stage (Fig. 8).

In the early blastocyst embryos, the nucleolusdisplays no more condensed areas and is heavilylabelled with the two antibodies. They both co-localizeon the DFC and GC, the FC being devoid of labelling(Fig. 9). The staining with silver gives the same resultsas described for the previous stage.

In all cases, the nucleoplasmic as well as thecytoplasmic labelling were low for both the antibodiesused.

In the control sections, incubated in the absence ofthe primary antibodies and, in the case of the anti-B23,also in the absence of the secondary antibody, thebackground was negligible.

Discussion

Our results on the immunolocalization of proteins B23

Fig. 7. Labelling with the anti-nucleolin antibody in morula. Thenucleolus is decondensing and onlyremnants of the compact mass are stillpresent (large arrows). These denseareas are not labelled, while theantibody strongly associates with thenucleolonema, consisting of DFC(small arrows) and GC (g). X28000.Fig. 8. Silver staining at morula stage.The residual compact areas(arrowheads) are weakly stained withsilver, while the DFC and an FC(arrow) are covered with precipitates.X30000.

and nucleolin and their correlation with silver stainingon mouse preimplantation embryos can be summarizedas follows:

(1) In all the stages where the compact core nucleolarmass is present, the immunolabelling can also beobserved on these condensed areas; however, theremnants of these areas at morula stage are notlabelled.

(2) The two antibodies co-localize in the DFC andGC of the nucleolus but they do not label FCs.

(3) Silver staining appears as a finely granularprecipitate on the compact nucleolar mass starting atthe 4-cell stage, while in the earlier stages these areasare virtually devoid of staining.

(4) Silver staining occurs in the FC and the DFC butnot in the GC.

Previous studies on nucleoli of proliferating cells inculture (Escande etal. 1985;Biggiogera etal. 1989) havedemonstrated that the pattern of silver staining corre-

1268 M. Biggiogera and others

'• - t

Fig. 9. Labelling with the anti-nucleolin antibody in earlyblastocyst. The nucleolus iscompletely deconsensed andboth the DFC and GC form astrongly labelled network. FCs(arrows) are not labelled.X21000.

sponds to the immunolocalization of proteins B23 andnucleolin only in the DFC. The FCs, although stronglysilver stained, remain unlabelled with either of the twoantibodies. In contrast, the GC is labelled with the twoantibodies but is devoid of silver deposits. Similarresults were obtained on mouse testis cells. Neither inspermatocyte nucleoli (active nucleoli) nor in earlyspermatid nucleoli (nucleoli undergoing inactivation)were we able to establish a direct relationship betweensilver staining and distribution of proteins B23 andnucleolin by immunolabelling (Biggiogera et al. 1990).

In the present work, we have shown that in nucleolarreactivation there is incomplete overlapping betweensilver staining and immunocytochemical detection ofB23 and nucleolin with regards to the differentnucleolar structural components. These proteins havebeen proposed as the major silver staining nucleolarconstituents (Lischwe et al. 1979). It has been reportedthat the silver staining mechanism involves the presenceof clusters of thiol groups (De Capoa et al. 1982). Onecan then speculate about the occurrence of sulfhydrylswithin the FC and DFC and their absence (or theiroxidation to form -S-S- bonds) within the GC. As wefailed to label the FC, it is probable that the silverstaining in this region is due to a constituent other thanB23 or nucleolin (Biggiogera et al. 1989). According toour results, these proteins are detectable only in theDFC and GC. The data on immunolocalization andsilver staining agree well as far as the DFC is concerned.On the contrary, the immunolabelling on the GC doesnot coincide with the silver reaction. It might well bethat in this nucleolar constituent, proteins B23 and/ornucleolin are present in a modified form, the epitopes of

which are still well recognizable by the correspondingantibody, but which is devoid of SH groups. This wouldaccount for the absence of silver staining. Although thisspeculation looks tempting, further experiments arenecessary in order to elucidate functional modificationstaking place at the level of the silver-stained nucleolarproteins.

As for the detection of the proteins B23 and nucleolinwith regards to the stage of embryogenesis and to thenucleolar reactivation, we have found the immuno-labelling on the dense core areas up to the 8-cell stage.The fragmentation of these areas taking place in morularesults in complete disappearance of labelling with bothantibodies within the compact core fibrillar residues.These same compact areas are, however, still finelystained with silver. In the reticular part emerging fromthese areas, the newly formed nucleolonema, consistingof usual nucleolar components, anti-B23 and anti-nucleolin antibodies occur on the DFC and GC but noton the FCs. As already mentioned, FCs and DFC arethe only nucleolar components stained with silver.

The two nucleolar phosphoproteins are detected asearly as within the prenucleolar bodies and appearmore abundant in the functional nucleoli in the courseof their reactivation, where most immunolabelling ispresent over the nucleolonema. At the 2-cell stage, thelabel due to the anti-B23 probe observed on theprenucleolar bodies appears much less abundant thanthat after the anti-nucleolin labelling. Whether thisphenomenon is related to a possible difference in thestability of these proteins or to a physiological changeoccurring during this developmental stage, remains tobe elucidated.

B23 and nucleolin in early mouse embryos 1269

The presence of the proteins B23 and nucleolin,detected by means of specific antibodies, has beenobserved in proliferating HeLa cell nucleoli (Biggiogeraet al. 1989), inactivating spermatid nucleoli (Biggiogeraet al. 1990) and re-activating embryonic nucleoli(present paper). These results, together with dataobtained from cell systems where nucleolar activity hasbeen experimentally reduced or arrested (Seegers et al.1985) or from mitotic cells (Ochs et al. 1983; Spector etal. 1984), show that the above proteins are detectedregardless of the level of nucleolar activity. In addition,it appears obvious from previous reports (Biggiogera etal. 1989; Escande et al. 1985; see also Fakan andHernandez-Verdun, 1986, for review) as well as fromour present results that silver staining is due only in partto these two proteins and that other substance(s) wouldbe responsible for nucleolar silver stainability. More-over, since silver staining also takes place in nucleolidevoid of transcriptional activity (Hernandez-Verdun etal. 1985; Dimova et al. 1982; Sanchez-Pina et al. 1984;Daskal et al. 1980; Escande-Geraud et al. 1985) or inmitotic NORs (Hernandez-Verdun etal. 1980; Ploton etal. 1987), it cannot be considered as a general marker ofnucleolar activity. Nevertheless, the increased densityof anti-B23 and anti-nucleolin labelling within nucleolarcomponents involved in rRNA synthesis and processingduring nucleolar embryonic reactivation, suggestssome, even though possibly indirect, involvement ofthese proteins in the pre-rRNP formation machinery. Inthis context, it is interesting to mention a recent reportspeculating about a possible role of these two proteinsin the transport of ribosomal precursors between thenucleus and the cytoplasm (Borer et al. 1989). In vitroassociation studies previously showed binding of nuc-leolin to the spacer sequence within cloned rDNA(Olson et al. 1983). Later experiments reported mostC23 co-sedimenting with rapidly labelled rRNAextracted from isolated nucleoli (Herrera and Olson,1986). Silver staining of spread ribosomal transcriptioncomplexes reveals, however, silver deposits predomi-nantly on the DNP axis of the gene, compared to theuntranscribed spacer regions or nascent RNP fibrils(Angelier etal. 1982). All these data together, althoughsuggestive with regards to a possible role for proteinsB23 and nucleolin in rRNA packaging, show thatfurther experimental evidence is necessary to clarify thefunctions of these proteins in the cell.

The authors would like to thank Drs M.-L. von Schack andV. Kopecny for critical reading of the manuscript, Ms F. FlachBiggiogera for technical assistance and Ms C. Cottier forphotographic work.

This work was supported by the Swiss National ScienceFoundation (31-26417.89).

M.B. is a recipient of a fellowship from the Fondation pourdes Bourses d'Etudes Italo-Suisse, Lausanne.

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(Accepted 10 September 1990)