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Analysis of nucleolar components by combined silver staining and serial sections procedures
Cell Biology
International
Reports,
Vol. 6, No. 7, July
659
1982
ANALYSISOF NUCLEOLAR COMPONENTS BY COMBINED SILVER STAINING ANDSERIAL SECTIONSPROCEDURES E. Cauve
Laboratorio Casilla Celular.
(I)
and
P. Eapomfa
(2)
Universidad de Microscopia Electrcnica. Chile (1) and lnstituto 130-V, Valparaiso, C.S. I-C. Vela’zquez 144, Madrid-6, Spain
de Valpara?so. de Biologia (‘2).
ABSTRACT The nucleolus in Sertoli cells, spermatogonia and spermatocytes of tibit testis was studied under electron microscopy using a silver staining method in combination with serial section reconstruction. Fibrillar centers CFc) as part of the nucleolar organizer region (NOR) are usually observed in these nucleoli and appear poorly stained after silver method which stain nucleolar acidic pro_ stains preferentially pa& of the dense fibrillar teins . Silver components of the nucleolus which appears like strings inside the nucleolar mass merging from part of the fibrillar center. The quantity and extent of the silver reaction seems to be correlated to the degree of transcriptional activity of each type of nucleolus. Moreover, the methods used allow the topography of the different nucleolar compxents to be inferred. Key
WU&&:
Nucleolus Electron
- Nucleolar microscopy.
organizer
-
Silver
staining
-
INTRODUCTION Studies by means of elecmn microscopy of the nucleolar strut ture have established a bowledge of their components in many cell types and during different events (Recher et al. 1969; Smetana and Busch, 1974; Gim&ez-Martin et al., 1977; Mirre and Stahl, 1978). Fibrillar and granular compnents are well known as elements related to the synthetic activities of this organelle. Recently the idea that a less dense and rounded structure, the "fibrillar center" corresponding to the ribosomal cistrons of chro matin, has been found associated with nucleolar fibrillar compnen7 which surrounds this element (Lepoint and Goessens, 1978; Goessens and Lepoint, 1979). The fibrillar center would correspond to the nucleolar organizer region or at least to a part of such chromesome region as mentioned in the studies previously quoted. For the cytological detection of the NOR and nucleolus, silver staining procedures have been employed using light (Goodpasture and Bloam, 1975) and electron microscopy (Hern&dez-Verdun et al. 1980; 0309-l
65ll82/070669-09l$03.00/0
@ 1982 Academic
Press Inc. (London)
Ltd.
660
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
KS.mer and Esponda, 1979). Da&al et al. (1980) show that under special conditions silver reacts with the acidic proteins of the fibrillar component of the nucleolus, which were previously identified as protein C23 and B23 (Da&al et al., 1974) and seems to be correlated with the transcriptional activity of the r-DNA (Da&al et al., 1980). These two phosphorylated nucleolar proteins, C23 and B23, are selectively stained with silver on polyacrylamide gels by the procedures,,used for silver staining of nucleolus (Lischwe et al., 1979). In.this paper we analyse with the silver staining method described by Daskal et al. (1980) different morphological types of nucleoli which appear in the testis of rabbit with the aim of obtaining new information and approaches to the interrelationships of nucleolar components. MATERIALAND METHODS Adultmale laboratoryrabbitwereused. Seminiferous tubules were obtained by surgical dissection and were treated as follows: a> For conventional procedures, fixation was carried out in 2% glutaraldehyde in cacodylate buffer at pH 7.2, postfixed in 0~04 1% in the same buffer), dehydrated in ethanol series and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. b) For silver staining the procedure described by Da&al et al. (1980) was used, employing seminiferous tubules fixed in Glutaraldehyde as above. Samples were postfixed in methanolacetic acid (3:l) for 5 min, rinsed in buffer and incubated with a 50%AgNO3 solution under an infrared light (Philips IR-25OW) for 10-15 min. After rinsing and postincubation in an equal mixture (1:l) of 100% AgNO3and formalin (37% w/v> under infrared light, tubules were dehydrated and embedded in Upon. Single hole grids covered with a film of Formvar were used for selected ribbons of These serial sections were stained sections about 100 rn-~thick. with aqueous uranyl acetate (2%). A Reichert (Cm-U2) ultrticrotome and a Zeiss EM 9 electron microscope were used. RESULTS These cells are identified by their size, a> Sp~atagonia.morphology and position in the tubules (see Roosen-Runge, 1977). Nuclei appear rounded or elongated in shape with the atin One, two or three nucleoli appear in single uniformly distributed. sections showing a partially rounded and campact morphology (Figs. land 3). Three components can be clearly seen in these nucleoli: a) A rounded shape and less stained body of about 0.3 um in diameteu,, ccmposed of tight packed fibres which seem to be the "fib) Dense strings which are ccmposed of fibres brillar center". (fibrillar ccmponent) with are located around the fibrillar center. c> A granular component (see Figs. l-2). After silver reaction the fibrillar center shows a slight reaction with respect to the fibrillar ccanponent, which is strongly stained
Cell Biology International
661
Reports, Vol. 6, No. 7, July 1982
Fig. 1 Nucleolus from a Spermatogonia. The different components can be observed. Fibri 1 lar center (Fc) ; f ibri 1 lar component (F) ; gr,anular component (G). Fi g. 2 At higher magnification zi Iassociated to chromatin (Ch)
a fibrillar and dense
center (Fc) is observ f ibril lar component (FT.
Fig. 3 Spermatogonial nucleus stained with silver, appear in a peripherical disposition. Silver grains located in strands delimited by nucleolar vacuoles Fig. 4 reaction
Silver reaction is located in
three nucleoli are selectively (arrows).
in a fibrillar center (Fc). The strong fibrillar component that-emerge from Fc. All bars indicate 0.5 urn.
662
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
Fig . 5 (a-f) Part of serial sections of a spermatogonial nucleolus The fibrillar center (arrows) can be clearly stained with s i lver. (b to e). A strong silver reaction appear as strings differentiated to nucleolar vacuoles. The fibrillar of grains in c lose relation Bar: 0.5 urn. react ion. center shows a sl ight
6 Schemat ic representation Fig. nucleolus from Fig. 5.
of
an entirety
serial
sections
of
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
663
are more clearly demonstrated (Figs. 3 and 4). These observations an example of which is shown in Figs. by serial sectioning analysis, This type of study allows the topographical location of 5 (a-f>. the silver reaction inside the nucleolar mass. The strongest reaction is distributed in strings-like masses closely related to a rounded structure which undoubtly represents the Fc. The diameter and positions of this strongly stained material seems to correspond to the fibrillar component observed in conventional stained sections (Figs. 5 and 6). Sertoli cells present the typical characb) Sgntuficti.mammalian species teristics described previously in different of (Fawcet-t. 1975). Nuclei appear elongated and with invaginations the nuclear envelope, with the chromatin uniformly distributed and showing only two or three dense chrcmatin masses. Only one nucleolus has been observed, ccanposed by many dense and elongated masses composed by fibres (fig. 7). Such masses seem to be preferentially and in which fibrillar centers can be observed (Fig. 7). When serial section reconstructions were made the interrelationships of the nucleolar masses could be observed (Fig. 8 a-c>. Silver staining and serial sections of such nucleoli show that silver grains are concentrated and packed in definite regions of the nucleolar mass, but occupy the main part of the nucleolar bulk in comparison with what can be observed in spermatogonia. c> Phimahg Ape4mdtacgXt2.- These cells are recognized by their position and size as well as their nuclear characteristics, as the presence of synaptonemal complexes during zigotene and pachytene stages (Roosen-Runge, 1977). %o or three nucleoli appear located close to the nuclear envelope and with a morphology similar to that of the Sertoli cells, but not as prcxninent as in the latter (see Fig. 9). When using the silver staining method a fine core of silver grains appears in the nucleolar masses as can be seen in Fig.10. DISCUSSION Two different nucleolar forms have been observed in the studied cells, a classical form of compact nucleolus in spermatogonial cell and an elongated or nucleolonemal-like nucleoli in Sertoli cells and spermatocytes. These two forms have similar components but are distributed in a different pattern. Cytogenetic studies on the number of NORs in the rabbit demonstrate the presence of three pairs of chromosomes which carry the nucleolar genes (Martin- De Leon, 1980). The nLnnber of nucleoli in spermatogonia show a correspondence with the NORs number, being the maximum number of nucleoli observed of three, each with their own Fc. The morphology of the unique Sertoli cell nucleolus seems to represent an aggregation of chromosomes carrying NORs, a fact which apparently appears represent ed by the close vicinity of the fibrillar centers (see Fig. 7). Thz Fc of the different nucleoli studied here show similar characteristics to those described previously in different materials (Goessens and Lepoint, 1979) being related to the perinucleolar chromatin and
Cell Biology
Cg. .! tibrlllar
A Sertol i cell nucleolus . _ . . material associated to
Fig. 8 (a-d) that of Fig. a fine string
f ibril
International
Part
Vol. 6, No. 7, July 1982
by many elongated centers (arrow).
masses
of
of
a serial section of a nucleolus similar to Silver grains are observed in with silver. disposition (arrows).
7 stained
A Primary Nucleoli lo-granular
Fig. 10 of silver
formed fibrillar
Reports,
A similar grains
spermatocyte are formed material. nucleolus are observed
with two nucleolus can be seen by numerous elongated masses of to Fig. 9 stained with arround each nucleolar All bars indicate
Strands silver. mass (arrows). 0.5 urn.
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
665
to the fibrillar ccxnponent. The studies of Da&al et al. (1974; 1980) mentioned above have clearly shown that the silver stained materialcorrespondto acidic proteins andthatthequantity of silver grains seems to be correlated with the degree of preriboscmal Moreover, silver staining of NORshas been correlatRNA synthesis. ed with nucleolar proteins C23 and B23 (Lischwe et al., 1979). Also, imnunological methods reveal that these two major nucleolar protein the nucleoli of normal and ins, are localized pred mtly tumour cells (Olson et al., 1981; Michalik et al., 1981). Recently, Lischwe and co-workers have shown that protein C23 is localized in the fibrillar centers within the nucleolus and have suggested that Fran the it could be involved in the riboscmal gene activity. point of view of the possible association of these proteins with synthetic activities silver grains could be a measure of the degree In this regard, we can observe that the number of such synthesis. of silver granules of the Fc is considerably smaller than in the fibrillar component. In this aspect scxneauthors claimed that the active r-DNA OCCUPSin the fibrillar region of the nucleolus (Granboulan and Granboulan, 1965; Daskal et al., 1974) and the Fc contained a non transcriptional NORDNA (Goessens and Lepoint, 1979). Onthe otherhand such proteins could play an important role in the maintenance of the nucleolar structure as a part of the nucleolar skeletal structures (for references on nucleolar skeleton see Franke et al., 1981). However, it TTEIE&LSto be proved the specific function of these nucleolar proteins. Finally, we can conclude that through serial section reconstructions: The number of silver grains are considerable in regions which seems to correspond to the fibrillar component. Nevertheless, the reaction does not occupy all these fibrillar strands, but only part of them as can be seen in Sertoli cells and spermatocytes . The application of serial section reconstruction gives the possibility of obtaining an idea of the disposition of such fibrillar ccanponent, and in this form makes it possible to represent the run over of the active loops of r-DNA inside the nucleolar mass. Apparently, the radial disposition of such loops from par-t of the Fc can be infered. This idea has been previously schematized for the FC in certain meiotic prophase stages of mouse oocytes (M.i.rre and Stahl, 1981). The quantification of the stained material is also possible after using the combination of the two methods, using different types of nucleoli as in the case of the present paper, or in nucleoli during different metabolic activities. In-this regard, our results show that the Sertoli cell nucleolus has considerable stained material in comparison with other cells, a fact that could be correlated to the higher activity which develops this nutritive and secretory cell (Fawcett, 1975). In comparison, the stained material in spermatocytes is scarce, which could be related to a decreased degree of r-RNA synthesis for such cells, .as was demonstrated for other mamnalian cells during meiotic prophase (Tres, 1975).
666
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
ACKNO-S We thanks to Fidel Vargas for technical assistence and Pilar Valenzuela for h@ help in the English correction. This mrk was partially supported by a grant N 344-795 from SDCACIUniversidad de Chile and a grant fmn the Cm&ion Asesora para la Investigaci& Cientifica y T&mica. Correspondence should be addressed to Dr. Eduardo Couve. REFERENCES
Da&al, Y.; Prestayko A.W. and Busch, H. (1974) Ultrastruc-tuml and biochemical studies of the isolated fibrillar ccxnponent of nucleoli frm Novikoff hepatcma ascites cells. Experimental Cell Research, 88, 1-14. Da&al, Y.; Smetana, K. and Busch, H. (1980) Evidence from studies on segregated nucleoli that nucleolar silvm staining proteins C23 and B23 are in the fibrillar ccqxment. Experimental Cell Research, 127, 285-291. Fawcett, D.W. (1975) Ultrastructure and function of the Sertoli cell. In Handbook of Physiology, Section 7, Vol. V, pp. 21-55. American Physiological Society. Washington. Fkanke, W.W.; Kleinschmidt, J.A.; Spring, H.; Kmhne, G.; Grund, C.; Trendele.nburg, M.F.; Stoehr, M. and Scheer, U. A nucleolar skeleton of protein filaments demstmted in amplified Journal of Cell Biology, 90, 289nucleoli of Xenopus laevis. 299 (1981). Gim&nez-Martin, G.; De la Terre, C.; tipez-sez, J.F. and Esponda, P. (1977) Plant nucleolus: Structure and Physioloa. Cytobiologie, 14, 421-462. Goessens, G. and Lepoint, A. (1979) The nucleolus organizing regions (NOR's): recent data and hypothesis. Biologie Cellulaire, 35, 211-220. Goodpasture, C. and Blcmn, S.E. (1975) Visualization of nucleolar organizer regions in n-mnnalian czhnmoscines u&-g silver chlxrnosoma, 53, 37-50. stairk-lg. Granboulan, N. and Granbmlan, P. (1965) Cytochmie ul-trastructuraExperimental Cell Research. 38, 604-619. le du nucl&ole. Hern&dez-Verdun, D.; Hubert, J.; Bourgeois, C. and Bouteille, M. (1980) Ultrastructmal localization of &NOR proteins in the nucleolus during the cell cycle and in other nucleolar ct-ranosama 79, 349-362. structures. tiimer, D.B. and Esponda, P. (1979) Nucleolar fibrillar centers in European Journal of Cell Biology, muse spermtid nucleoli. 20, 156-158. Lqoint, A. and Goessens, G. (1978) Nucleologenesis in Enrlich Experimental Cell Research, 117, 89-94. tumow? cells.
Cell Biology
International
Reports,
667
Vol. 6, No. 7, July 1982
Lischwe, M.A.; Smetana, K.; Olson, M.O.J. and Busch, H. (1979) Proteins C23 and B23 are the major nucleolar silver staining Life Sciences, 25, 701-708. proteins. Lischwe, M.A.; Richards, R.L.; Busch, R.K. and Busch, H. (1981) Localization of phosphoprotein C23 to nucleolar structures and to nucleolus organizer regions. Experimental Cell Research, 136, 101-109. Martin-De Leon, P.A. (1980) Location of the18S and 28s rRNA cistrons in the gencme of the domestic rabbit (Oryctolagus cuniculus, L.). Cytogenetics and Cell Genetics, 28, 34-40. Michalik,J.;Yeoman, L.C. and Busch, H. (1981) Nucleolar localization of protein B23 (37/5.1) by irmnunocytochemical Life Sciences, 28, 1371-1379. techniques. Mirre, C. and Stahl, A (1978) Ultrastructure and Activity of the nucleolar organizer in the mouse oocyte during meiotic prophase. Journal of Cell Science. 31, 79-100. Mirre, C. and Stahl, A. (1981) Ultrastructural organization, sites of trenscription and distribution of fibrillar centers in the nucleolus of the mouse oocyte. Journal of Cell Science, 48, 105-126. Olson, M.O.J.; Guetzow, K. and Busch, H. (1981) Localization of phosphoIx&xin C23 in nucleoli by immunological methods. Experimental Cell Research, 135, 259-265. Recher, L.; Whitescarver, J. and Briggs, L. (1969) The fine structure of a nucleolar constituent. Journal of Ultrastructure Research, 29, 1-14. Roosen-Runge, E.C. (1977) The process of spexmatogenesis in animals. Cambridge University Press, Cambridge. Smetana, K. and Busch, H. (1974) The nucleolus and nucleolar DNA. In The Cell Nucleus (H. Busch Ed.) Vol. I, pp. 73-147. &d&c Press, New York. Tres, L.L. (1975) Nucleolar RNA synthesis of meiotic prophase spermatocytes in the Humantestis. chromosama, 53, 141-151.
Received:
11th January
1982
Revised version 5th April 1982
accepted:
International
Reports,
Vol. 6, No. 7, July
659
1982
ANALYSISOF NUCLEOLAR COMPONENTS BY COMBINED SILVER STAINING ANDSERIAL SECTIONSPROCEDURES E. Cauve
Laboratorio Casilla Celular.
(I)
and
P. Eapomfa
(2)
Universidad de Microscopia Electrcnica. Chile (1) and lnstituto 130-V, Valparaiso, C.S. I-C. Vela’zquez 144, Madrid-6, Spain
de Valpara?so. de Biologia (‘2).
ABSTRACT The nucleolus in Sertoli cells, spermatogonia and spermatocytes of tibit testis was studied under electron microscopy using a silver staining method in combination with serial section reconstruction. Fibrillar centers CFc) as part of the nucleolar organizer region (NOR) are usually observed in these nucleoli and appear poorly stained after silver method which stain nucleolar acidic pro_ stains preferentially pa& of the dense fibrillar teins . Silver components of the nucleolus which appears like strings inside the nucleolar mass merging from part of the fibrillar center. The quantity and extent of the silver reaction seems to be correlated to the degree of transcriptional activity of each type of nucleolus. Moreover, the methods used allow the topography of the different nucleolar compxents to be inferred. Key
WU&&:
Nucleolus Electron
- Nucleolar microscopy.
organizer
-
Silver
staining
-
INTRODUCTION Studies by means of elecmn microscopy of the nucleolar strut ture have established a bowledge of their components in many cell types and during different events (Recher et al. 1969; Smetana and Busch, 1974; Gim&ez-Martin et al., 1977; Mirre and Stahl, 1978). Fibrillar and granular compnents are well known as elements related to the synthetic activities of this organelle. Recently the idea that a less dense and rounded structure, the "fibrillar center" corresponding to the ribosomal cistrons of chro matin, has been found associated with nucleolar fibrillar compnen7 which surrounds this element (Lepoint and Goessens, 1978; Goessens and Lepoint, 1979). The fibrillar center would correspond to the nucleolar organizer region or at least to a part of such chromesome region as mentioned in the studies previously quoted. For the cytological detection of the NOR and nucleolus, silver staining procedures have been employed using light (Goodpasture and Bloam, 1975) and electron microscopy (Hern&dez-Verdun et al. 1980; 0309-l
65ll82/070669-09l$03.00/0
@ 1982 Academic
Press Inc. (London)
Ltd.
660
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
KS.mer and Esponda, 1979). Da&al et al. (1980) show that under special conditions silver reacts with the acidic proteins of the fibrillar component of the nucleolus, which were previously identified as protein C23 and B23 (Da&al et al., 1974) and seems to be correlated with the transcriptional activity of the r-DNA (Da&al et al., 1980). These two phosphorylated nucleolar proteins, C23 and B23, are selectively stained with silver on polyacrylamide gels by the procedures,,used for silver staining of nucleolus (Lischwe et al., 1979). In.this paper we analyse with the silver staining method described by Daskal et al. (1980) different morphological types of nucleoli which appear in the testis of rabbit with the aim of obtaining new information and approaches to the interrelationships of nucleolar components. MATERIALAND METHODS Adultmale laboratoryrabbitwereused. Seminiferous tubules were obtained by surgical dissection and were treated as follows: a> For conventional procedures, fixation was carried out in 2% glutaraldehyde in cacodylate buffer at pH 7.2, postfixed in 0~04 1% in the same buffer), dehydrated in ethanol series and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate. b) For silver staining the procedure described by Da&al et al. (1980) was used, employing seminiferous tubules fixed in Glutaraldehyde as above. Samples were postfixed in methanolacetic acid (3:l) for 5 min, rinsed in buffer and incubated with a 50%AgNO3 solution under an infrared light (Philips IR-25OW) for 10-15 min. After rinsing and postincubation in an equal mixture (1:l) of 100% AgNO3and formalin (37% w/v> under infrared light, tubules were dehydrated and embedded in Upon. Single hole grids covered with a film of Formvar were used for selected ribbons of These serial sections were stained sections about 100 rn-~thick. with aqueous uranyl acetate (2%). A Reichert (Cm-U2) ultrticrotome and a Zeiss EM 9 electron microscope were used. RESULTS These cells are identified by their size, a> Sp~atagonia.morphology and position in the tubules (see Roosen-Runge, 1977). Nuclei appear rounded or elongated in shape with the atin One, two or three nucleoli appear in single uniformly distributed. sections showing a partially rounded and campact morphology (Figs. land 3). Three components can be clearly seen in these nucleoli: a) A rounded shape and less stained body of about 0.3 um in diameteu,, ccmposed of tight packed fibres which seem to be the "fib) Dense strings which are ccmposed of fibres brillar center". (fibrillar ccmponent) with are located around the fibrillar center. c> A granular component (see Figs. l-2). After silver reaction the fibrillar center shows a slight reaction with respect to the fibrillar ccanponent, which is strongly stained
Cell Biology International
661
Reports, Vol. 6, No. 7, July 1982
Fig. 1 Nucleolus from a Spermatogonia. The different components can be observed. Fibri 1 lar center (Fc) ; f ibri 1 lar component (F) ; gr,anular component (G). Fi g. 2 At higher magnification zi Iassociated to chromatin (Ch)
a fibrillar and dense
center (Fc) is observ f ibril lar component (FT.
Fig. 3 Spermatogonial nucleus stained with silver, appear in a peripherical disposition. Silver grains located in strands delimited by nucleolar vacuoles Fig. 4 reaction
Silver reaction is located in
three nucleoli are selectively (arrows).
in a fibrillar center (Fc). The strong fibrillar component that-emerge from Fc. All bars indicate 0.5 urn.
662
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
Fig . 5 (a-f) Part of serial sections of a spermatogonial nucleolus The fibrillar center (arrows) can be clearly stained with s i lver. (b to e). A strong silver reaction appear as strings differentiated to nucleolar vacuoles. The fibrillar of grains in c lose relation Bar: 0.5 urn. react ion. center shows a sl ight
6 Schemat ic representation Fig. nucleolus from Fig. 5.
of
an entirety
serial
sections
of
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
663
are more clearly demonstrated (Figs. 3 and 4). These observations an example of which is shown in Figs. by serial sectioning analysis, This type of study allows the topographical location of 5 (a-f>. the silver reaction inside the nucleolar mass. The strongest reaction is distributed in strings-like masses closely related to a rounded structure which undoubtly represents the Fc. The diameter and positions of this strongly stained material seems to correspond to the fibrillar component observed in conventional stained sections (Figs. 5 and 6). Sertoli cells present the typical characb) Sgntuficti.mammalian species teristics described previously in different of (Fawcet-t. 1975). Nuclei appear elongated and with invaginations the nuclear envelope, with the chromatin uniformly distributed and showing only two or three dense chrcmatin masses. Only one nucleolus has been observed, ccanposed by many dense and elongated masses composed by fibres (fig. 7). Such masses seem to be preferentially and in which fibrillar centers can be observed (Fig. 7). When serial section reconstructions were made the interrelationships of the nucleolar masses could be observed (Fig. 8 a-c>. Silver staining and serial sections of such nucleoli show that silver grains are concentrated and packed in definite regions of the nucleolar mass, but occupy the main part of the nucleolar bulk in comparison with what can be observed in spermatogonia. c> Phimahg Ape4mdtacgXt2.- These cells are recognized by their position and size as well as their nuclear characteristics, as the presence of synaptonemal complexes during zigotene and pachytene stages (Roosen-Runge, 1977). %o or three nucleoli appear located close to the nuclear envelope and with a morphology similar to that of the Sertoli cells, but not as prcxninent as in the latter (see Fig. 9). When using the silver staining method a fine core of silver grains appears in the nucleolar masses as can be seen in Fig.10. DISCUSSION Two different nucleolar forms have been observed in the studied cells, a classical form of compact nucleolus in spermatogonial cell and an elongated or nucleolonemal-like nucleoli in Sertoli cells and spermatocytes. These two forms have similar components but are distributed in a different pattern. Cytogenetic studies on the number of NORs in the rabbit demonstrate the presence of three pairs of chromosomes which carry the nucleolar genes (Martin- De Leon, 1980). The nLnnber of nucleoli in spermatogonia show a correspondence with the NORs number, being the maximum number of nucleoli observed of three, each with their own Fc. The morphology of the unique Sertoli cell nucleolus seems to represent an aggregation of chromosomes carrying NORs, a fact which apparently appears represent ed by the close vicinity of the fibrillar centers (see Fig. 7). Thz Fc of the different nucleoli studied here show similar characteristics to those described previously in different materials (Goessens and Lepoint, 1979) being related to the perinucleolar chromatin and
Cell Biology
Cg. .! tibrlllar
A Sertol i cell nucleolus . _ . . material associated to
Fig. 8 (a-d) that of Fig. a fine string
f ibril
International
Part
Vol. 6, No. 7, July 1982
by many elongated centers (arrow).
masses
of
of
a serial section of a nucleolus similar to Silver grains are observed in with silver. disposition (arrows).
7 stained
A Primary Nucleoli lo-granular
Fig. 10 of silver
formed fibrillar
Reports,
A similar grains
spermatocyte are formed material. nucleolus are observed
with two nucleolus can be seen by numerous elongated masses of to Fig. 9 stained with arround each nucleolar All bars indicate
Strands silver. mass (arrows). 0.5 urn.
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
665
to the fibrillar ccxnponent. The studies of Da&al et al. (1974; 1980) mentioned above have clearly shown that the silver stained materialcorrespondto acidic proteins andthatthequantity of silver grains seems to be correlated with the degree of preriboscmal Moreover, silver staining of NORshas been correlatRNA synthesis. ed with nucleolar proteins C23 and B23 (Lischwe et al., 1979). Also, imnunological methods reveal that these two major nucleolar protein the nucleoli of normal and ins, are localized pred mtly tumour cells (Olson et al., 1981; Michalik et al., 1981). Recently, Lischwe and co-workers have shown that protein C23 is localized in the fibrillar centers within the nucleolus and have suggested that Fran the it could be involved in the riboscmal gene activity. point of view of the possible association of these proteins with synthetic activities silver grains could be a measure of the degree In this regard, we can observe that the number of such synthesis. of silver granules of the Fc is considerably smaller than in the fibrillar component. In this aspect scxneauthors claimed that the active r-DNA OCCUPSin the fibrillar region of the nucleolus (Granboulan and Granboulan, 1965; Daskal et al., 1974) and the Fc contained a non transcriptional NORDNA (Goessens and Lepoint, 1979). Onthe otherhand such proteins could play an important role in the maintenance of the nucleolar structure as a part of the nucleolar skeletal structures (for references on nucleolar skeleton see Franke et al., 1981). However, it TTEIE&LSto be proved the specific function of these nucleolar proteins. Finally, we can conclude that through serial section reconstructions: The number of silver grains are considerable in regions which seems to correspond to the fibrillar component. Nevertheless, the reaction does not occupy all these fibrillar strands, but only part of them as can be seen in Sertoli cells and spermatocytes . The application of serial section reconstruction gives the possibility of obtaining an idea of the disposition of such fibrillar ccanponent, and in this form makes it possible to represent the run over of the active loops of r-DNA inside the nucleolar mass. Apparently, the radial disposition of such loops from par-t of the Fc can be infered. This idea has been previously schematized for the FC in certain meiotic prophase stages of mouse oocytes (M.i.rre and Stahl, 1981). The quantification of the stained material is also possible after using the combination of the two methods, using different types of nucleoli as in the case of the present paper, or in nucleoli during different metabolic activities. In-this regard, our results show that the Sertoli cell nucleolus has considerable stained material in comparison with other cells, a fact that could be correlated to the higher activity which develops this nutritive and secretory cell (Fawcett, 1975). In comparison, the stained material in spermatocytes is scarce, which could be related to a decreased degree of r-RNA synthesis for such cells, .as was demonstrated for other mamnalian cells during meiotic prophase (Tres, 1975).
666
Cell Biology
International
Reports,
Vol. 6, No. 7, July 1982
ACKNO-S We thanks to Fidel Vargas for technical assistence and Pilar Valenzuela for h@ help in the English correction. This mrk was partially supported by a grant N 344-795 from SDCACIUniversidad de Chile and a grant fmn the Cm&ion Asesora para la Investigaci& Cientifica y T&mica. Correspondence should be addressed to Dr. Eduardo Couve. REFERENCES
Da&al, Y.; Prestayko A.W. and Busch, H. (1974) Ultrastruc-tuml and biochemical studies of the isolated fibrillar ccxnponent of nucleoli frm Novikoff hepatcma ascites cells. Experimental Cell Research, 88, 1-14. Da&al, Y.; Smetana, K. and Busch, H. (1980) Evidence from studies on segregated nucleoli that nucleolar silvm staining proteins C23 and B23 are in the fibrillar ccqxment. Experimental Cell Research, 127, 285-291. Fawcett, D.W. (1975) Ultrastructure and function of the Sertoli cell. In Handbook of Physiology, Section 7, Vol. V, pp. 21-55. American Physiological Society. Washington. Fkanke, W.W.; Kleinschmidt, J.A.; Spring, H.; Kmhne, G.; Grund, C.; Trendele.nburg, M.F.; Stoehr, M. and Scheer, U. A nucleolar skeleton of protein filaments demstmted in amplified Journal of Cell Biology, 90, 289nucleoli of Xenopus laevis. 299 (1981). Gim&nez-Martin, G.; De la Terre, C.; tipez-sez, J.F. and Esponda, P. (1977) Plant nucleolus: Structure and Physioloa. Cytobiologie, 14, 421-462. Goessens, G. and Lepoint, A. (1979) The nucleolus organizing regions (NOR's): recent data and hypothesis. Biologie Cellulaire, 35, 211-220. Goodpasture, C. and Blcmn, S.E. (1975) Visualization of nucleolar organizer regions in n-mnnalian czhnmoscines u&-g silver chlxrnosoma, 53, 37-50. stairk-lg. Granboulan, N. and Granbmlan, P. (1965) Cytochmie ul-trastructuraExperimental Cell Research. 38, 604-619. le du nucl&ole. Hern&dez-Verdun, D.; Hubert, J.; Bourgeois, C. and Bouteille, M. (1980) Ultrastructmal localization of &NOR proteins in the nucleolus during the cell cycle and in other nucleolar ct-ranosama 79, 349-362. structures. tiimer, D.B. and Esponda, P. (1979) Nucleolar fibrillar centers in European Journal of Cell Biology, muse spermtid nucleoli. 20, 156-158. Lqoint, A. and Goessens, G. (1978) Nucleologenesis in Enrlich Experimental Cell Research, 117, 89-94. tumow? cells.
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Received:
11th January
1982
Revised version 5th April 1982
accepted: