MOLECULAR REPRODUCTION AND DEVELOPMENT 56:198–206 (2000)
New Site of Temporary Storage of rRNA in O. mykiss Previtellogenic Oocytes S. FUSCO, S. FILOSA, M. IACOVIELLO, P. INDOLFI, S. TAMMARO, AND C.M. MOTTA* Department of Evolutive and Comparative Biology, University of Naples Federico II, Naples, Italy
ABSTRACT This article describes a new organelle found in the cytoplasm of the growth stage fish oocytes. In particular, we describe its organization at the morphological level and investigate its composition by different cytochemical and immunocytochemical approaches with both light and electron microscope. The conclusion is that the body is a peculiar protein scaffold functioning as a temporary trap for the storage of rRNA in the mid to late growth stage oocytes. Its presence would be related to the reorganization of the mass of amplified rDNA in micronucleoli and to the consequent temporary stop in the rRNA synthesis. Mol. Reprod. Dev. 56:198–206, 2000. r 2000 Wiley-Liss, Inc.
standard procedures using as primary antibodies a mouse anti-vimentin (Sigma, 1:50 dilution) and a mouse anti-actin (Sigma, 1:100 dilution). Both were revealed with a secondary antibody labeled with peroxydase (Sigma, 1:200 dilution). Nick translation in situ was performed according to Gold et al. (1993) using a DNA polimerase of E. coli and dNTP-digoxygenated from Boehringer. In situ rDNA hybridization was carried out according to Lehmann and Tautz (1994). The probe, obtained from C. hamatus, was kindly donated by Dr. Luisella Carratu` and labeled with the technical assistance of Dr. Rosaria Scudiero at the Istituto di Biochimica delle Proteine ed Enzimologia, CNR, Naples.
Key Words: rRNA; storage; oocytes; trout
Electron Microscopy and Immunocytochemistry Samples for morphological observations were fixed according to Karnowsky (1965), dehydrated in ethanol, and embedded in Epon 812 following the manufacturer’s instructions. Ultrathin sections were doublestained with uranyl acetate and lead citrate and observed under a model 301 Phillips electron microscope. Samples for immunocytochemical revelation of the DNA were prepared according to Bohrmann and Kellenberger (1994) using a mouse primary antibody (Boehringer, 1:8 dilution) and a secondary antibody labeled with colloidal gold (10 nm, Sigma, 1:20 dilution). Samples for RNAse-gold were prepared according to Bendayan (1981, 1982) and incubated 1 hr with 2 µl of the solution containing the enzyme (ICN, Milan, I, 10 µg/ml). Sections pretreated with pronase (10 mg/ml), DNAse (10 mg/ml) and RNAse (10 mg/ml) were also prepared and used as controls.
INTRODUCTION In the oocyte cytoplasm of several species of fishes we have reported the presence of peculiar round bodies whose structure and function remain unclear (Fusco et al., 1997). Their affinity for nucleolar stains suggested that these bodies might contain proteins and RNA and might therefore be involved in the storage of nutritional and/or informational materials during oogenesis. The aim of the present work was, therefore, to determine the organization and composition of these bodies by transmission electron microscopy (TEM) and immunocytochemical analyses, in the trout Oncorhynchus mykiss, in order to clarify their function during oocyte growth. MATERIALS AND METHODS Light Microscopy and Immunocytochemistry Ovaries dissected from animals obtained from commercial sources were cut into pieces and processed for light microscopy according to the following protocols. Materials were fixed in Bouin’s fixative for 6–12 hr, dehydrated, and embedded in wax according to standard procedures. Slides were then stained with haematoxylin-eosin, DAPI, distamycin, pironin, or PAS (Mazzi, 1977) while silver staining was performed according to Derenzini et al. (1992). Immunocytochemistry for DNA was carried out according to Moussa et al. (1994) using a mouse anti-DNA antibody (Boehringer, Milan, I, 1:2 dilution) revealed by a secondary antibody labeled with peroxydase (Sigma, Milan, I, 1:200 dilution). Staining for vimentin and actin were carried out following
r 2000 WILEY-LISS, INC.
In Vitro Labeling With Tritiated Uridine Pieces of ovary were maintained in vitro for 48 hr in a medium supplemented with 10% fetal cow serum and 20 µCi/ml tritiated uridine (Amersham International, Milan, I; S.A. 25–30 µCi/mM). Samples were then processed for light microscopy as previously described. Slides were coated with liquid nuclear emulsion (L2,
Grant sponsor: MURST, Progetto Nazionale entitled ‘‘Biologia ed Evoluzione del Riconoscimento e delle Interazioni nelle Cellule Animali.’’ *Correspondence to: Chiara Motta, Dip. Biologia Evolutiva e Comparata, via Mezzocannone 8, 80134 Napoli, Italy. E-mail:
[email protected] Received 25 October 1999; Accepted 27 December 1999
rRNA STORAGE IN TROUT OOCYTES Ilford, Milan, I), let dry and exposed at 4°C for about 4 weeks. Slides were then developed in Hypam and Phenisol according to the manufacturer’s instructions, and stained with haematoxylin-eosin. Image Acquisition and Processing Images from slides were acquired using a Progress 3008 camera (Kontron Elektronik, Eching, Germany) mounted on a Axioskop microscope (Zeiss, Milan, I) and a Kontron KS300 program. TEM images were printed on paper and acquired by scanner. Tables were assembled, lettered and numbered by using a commercial graphic program for PC and printed on high resolution Kodak paper. RESULTS Morphological Characterization of the Round Bodies At the light microscope the bodies appear clearly recognizable in the cytoplasm of the oocytes in the early primary growth stage (oocyte about 150 µm in diameter). The bodies are usually small (1–2 µm), present in a number of 2 to 6, and dispersed in the cytoplasm (Fig. 1a), mostly in the perinuclear region. In larger oocytes (300 µm diameter), in the mid-growth stage, the presence of one, or more rarely two bodies, ranging between 6–12 µm is noticed in all the oocytes. These bodies are located very close to the nuclear envelope (Fig. 1b), and often appear to be enclosed in the Balbiani body (Fig. 1c). By the late growth stage (oocytes 400–500 µm diam.), the round bodies have moved to the oocyte periphery (Fig. 1d) and start to dissolve becoming progressively smaller and irregular in shape (Fig. 1e). Dissolution occurs at about the same time the vitelline Balbiani body disperses in the cytoplasm forming a series of dense aggregates (Fig. 1f) that eventually disappear without leaving any apparent residue during the following cortical alveoli stage. At the electron microscope the round body shows a peculiar organization: it appears as a tridimensional net, not limited by any envelope, with cords regularly arranged so to form approximately polygonal grids (Fig. 2a and b). In the inner portion the cords are dense and finely granular; in the outer portion, by contrast, they appear more fibrillar (Fig. 2b and also Fig. 4g,h). Among cords, a typical cytoplasm containing a few small vesicles and dense aggregates together with occasional small mitochondria can be observed (Fig. 2b). The organization of the round body is very different to that of the nucleoli: these, in fact, appear large, round, and compact, with a high electron density and a finely fibrillogranular texture (Fig. 2c); during the growth stage they are placed very close to the nuclear envelope (Figs. 1b,c, 2c). Small aggregates, similar in density to the material forming the cords, can also be observed close to the envelope, in the cytoplasm (Fig. 2d). In the late growth stage oocytes, the round bodies under dissolution show a still clearly recognizable organization though the cords are mostly interrupted (Fig. 2e). These bodies are always found in the outer region of the cytoplasm, often very close to the chorion.
199
Biochemical Characterization of the Round Bodies Following treatment with proteinases the bodies undergo dissolution with the acquisition, at the TEM, of an ultrastructure that closely mimics that observed during dissolution under physiological conditions (compare Fig. 3a with Fig. 2e). In addition, bodies in early to mid growth stage show a high affinity for silver salts at both light (Fig. 3c) and electron microscope as demonstrated by the presence of a granular precipitate in correspondence of the cords but not of the surrounding portions of the cytoplasm (Fig. 3e,f). A significant staining of the body also is present following the reaction with the anti-actin (Fig. 3g) and the antivimentin antibodies (Fig. 3h) while, by contrast, no staining is observed following the PAS reaction (Fig. 3b). The affinity for the previously mentioned protein stains appears highly reduced or completely absent in bodies undergoing dissolution in the late growth stage (see Fig. 2d for example). Investigations carried out to determine the presence of nucleic acids in the round bodies indicate a significant positivity to pyronin (Fig. 4a), file DAPI (Fig. 4b), and distamycin (Fig. 4c) stains. The anti-DNA antibody, however, does not stain the body in either light (Fig. 4e) or electron microscope sections (Fig. 4f) even though the affinity of the antibody for this nucleic acid is clearly proven by the staining of the nuclear materials of both oocyte and follicle cells (Fig. 4e and 4e inset). The absence of a significant amount of DNA in the bodies is confirmed by the absence of staining following the in situ nick-translation reaction (Fig. 4d). Treatment of the ultrathin sections with an RNAsegold complex reveals the presence of significant labeling over the cords of the round body, in particular over the external, fibrillar portions (Fig. 4g,h). Here, the gold particles are at least fivefold more concentrated than over the rest of the cytoplasm, which also appears significantly labeled with respect to the background. Gold particles also are scattered over the oocyte nucleus and over follicle cells cytoplasm and nucleus. The presence of RNA in the round bodies is confirmed by the results of the in situ hybridization. The labeled cDNA probe for 18S RNA, in fact, specifically binds to the body as well as to the oocyte nucleoli (Fig. 4i). Finally, following in vitro treatment for 48 hr with tritiated uridine a significant labeling is observed over the nuclei and cytoplasms of most follicle cells (Fig. 4l). In early growth stage oocytes (Fig. 4l) labeling is noticed over the nucleoplasm, the nucleoli, and also over the cytoplasm. Labeling over small round bodies cannot be ascertained at this stage due to the fact that their average size (1 µm) is approximately comparable to that of the silver grains formed during autoradiography. In mid to late growth stage oocytes, labeling is located mainly over the nucleoplasm, while the nucleoli, the cytoplasm, and the round bodies are unlabeled (Fig. 4m).
200
S. FUSCO ET AL.
Fig. 1. Morphological characterization of the round bodies. (a) Group of early growing oocytes. The presence in the cytoplasm of small round bodies (arrow) can be noticed. N: nucleus. (b) Detail of the nucleus of a mid growth stage oocyte. A round body (arrow) is present in the cytoplasm, near the nuclear envelope (NE). The nucleoli (arrowhead) are also located close to the envelope, in the nucleoplasm. (c) Mid growth stage oocyte with the Balbiani body (BB) enclosing a round body (arrow). (d) Late growth stage oocyte showing a large round body (arrow) in the outer cytoplasm, close to the oocyte membrane. (e) Detail of a late growth stage oocyte with a dissolving round body (arrow): it appears irregular in shape and less dense. Notice the presence of the first cortical alveoli (CA) in the cytoplasm. (f) Growth stage oocyte showing a Balbiani body (BB) starting dissolution. Haematoxylin-eosin stain; a,c,d,f: 3350; b,e: 3800.
DISCUSSION The large cytoplasmic body present in growth stage oocytes of O. mykiss shows a rather peculiar morphology not resembling that shown by any other known cell structure. At the light microscope it appears as a dense
mass but at the TEM it reveals an organization in cords regularly arranged tridimensionally to form a sort of ‘‘bouquet.’’ This regular disposition, with almost polygonal grids, suggests that the assembly of the cords might be due, at least partly, to crystalline forces.
rRNA STORAGE IN TROUT OOCYTES
Fig. 2. Ultrastructural characterization of the round body. (a) Low magnification of a round body. It appears as a bouquet of dense cords in a cytoplasm rich in vesicles (*) and organelles. (b) Higher magnification showing a detail of the body. Cords are dense, homogeneous, finely granular with irregular, rather fibrillar edges, and form polygonal grids. Cytoplasm among cords contains dense aggregates (arrow), vesicles (*) and small mitochondria (M). (c) Detail of a mid growth stage oocyte. Notice the dense and compact nucleolus (nu) close to the
201
nuclear envelope (NE), and the presence of pores. The cytoplasm shows the Balbiani body (BB), a region enriched in mitochondria and small vesicles. (d) Detail of an early growth stage oocyte. Close to the nuclear envelope (NE), in the cytoplasm (Cy) several dense aggregates (arrow) can be observed. N: nucleus. (e) Round body under dissolution in a late growth stage oocyte. Most of the cords are already interrupted while the presence of polygonal grids (arrow) is still evident. a: 35,500; b: 335,000; c: 34,500; d: 322,000; e: 330,000.
202
S. FUSCO ET AL.
Fig. 3. Biochemical characterization of the round body: protein content. (a) TEM micrograph of an ultrathin section of a round body treated with proteinase. The body appears partly dissolved with cords interrupted and remnant of polygonal grids (arrow): this morphology closely resembles that observed in round bodies undergoing physiological resorbtion (compare with Fig. 2e). (b) Mid growth stage oocyte following a PAS reaction. The round body (arrow) is unstained, while a positivity to the reaction is observed over the chorion (C) and the glucidic droplets present in the outer cytoplasm of larger oocytes (*). N: nucleus. (c) Mid growth stage oocyte following silver staining. The round body (arrow) and the nucleoli (arrowheads) appear highly stained. (d) Late growth stage oocyte following silver staining. The
round body under dissolution (arrow) shows no affinity for silver salts as compared with the oocyte nucleoli (arrowheads). (e, f) Silver staining of ultrathin sections of a round body. Silver salts form electron-dense granular deposits over the cords (arrows) arranged to form polygonal grids. (g) Mid growth stage oocyte treated with the anti-actin antibody. A significant positivity is observed over the round body (arrow) and, to a lesser extent, over the nucleoplasm and cytoplasm of the small oocytes (*). (h) Mid growth stage oocyte treated with the anti-vimentin antibody. A significant positivity is observed over the round body (arrow). a: 315,000; b,c,g: 3350; d: 3800; e: 318,000; f: 335,000; g: 3450.
rRNA STORAGE IN TROUT OOCYTES The presence of such a structure in the cytoplasm of mid growth stage trout oocytes has been reported previously by Beams and Kessel in a paper published in 1973. In this case the authors acknowledged its presence without giving any details on its composition or function. In an our previous paper, at the light microscope, we report that round bodies also are present in many other species of fishes of either temperate (Oblada melanura), subarctic (Myoxocephalus scorpius), or antarctic (Trematomus bernacchii, T. hansoni) seas (Fusco et al., 1997). In the only antarctic species (Trematomus bernacchi) examined at the TEM, however, the body appeared different from the body described in the trout (Fusco et al., 1997). The meaning of such differences in organization is unclear. The origin of the round bodies also is not clear. The presence of several 1-µm bodies only in the early growing oocytes suggests that these may fuse together forming the larger body seen in the mid to late growing oocytes. This hypothesis, however, needs to be verified. As far as the composition is concerned, the cytochemical observations indicate that cords are made of proteins, in particular by nonglycosylated, acidic proteins. In fact no staining is obtained following the PAS reaction that, by contrast, stains the chorion and the glucid droplets present in the outer cytoplasm. The acidic nature of the proteins is demonstrated by their high affinity for silver salts (Hartung et al., 1983) and by their positivity to the anti-actin antibody: actin, in fact, is rich in charged residues, necessary for myosin binding (Miller and Reisler, 1995). Which form of actin is present is not known: the antibody, in fact, does not discriminate among the different isoforms but recognizes an epitope of 11 aa at the highly conserved, C-terminal end of the protein. The positivity to the anti-vimentin and to the silver salts (reported to stain specifically several nucleolar proteins; HernandezVerdun, 1982) indicates that the body has a very complex protein composition. Besides proteins, the body contains also significant amounts of nucleic acids: AT and GC bases in fact, are present as demonstrated by the positivity to specific stains such as DAPI and distamycin. Immunocytochemical data reveal that DNA is not present: no affinity is observed for the antibody at either light or electron microscope and, as a confirmation, no stains are obtained following the nick-translation reaction. The body, however, does contain RNA: it has a high affinity for pyronin and is significantly decorated by colloidal gold following treatment with the RNAse-gold complex. This technique reveals that the RNA is not homogeneously distributed over the cords but concentrated over the fibrillar edges. At this level, in fact, the number of gold particles increases significantly with respect to all the other regions of the nucleus and cytoplasm where RNA is also aboundant. As far as the nature of the RNA is concerned, the positivity to the in situ hybridization clearly indicates the presence of 18S rRNA. Preliminary data (not shown) also indicate the presence of 28S rRNA while the
203
presence of other RNAs, either ribosomal or messenger, cannot be excluded and, at the moment, is under investigation. The form in which the rRNA is stored is not clear: however, an organization in ribosome/ preribosome particles appears highly improbable since the edges of the cords, where RNA is concentrated, have a clearly fibrillar organization. The experiments with tritiated uridine indicate that the rRNA stored is synthesized during the early growth stage; in mid and late stages the nucleoli are unlabeled. The RNAs would then be released during the dissolution stage, concomitant with the resorbtion of the protein cords as suggested by the gradual loss of affinity for the different stains. The round body, therefore, would be a temporary trap for the ribosomal RNAs. It is noteworthy to consider that the assembly and dissolution of the round body occur at the same time the Balbiani body is also assembled and resorbed (Begovac and Wallace, 1988). It may be a coincidence or, most probably, be indicative of a change in the synthetic activities in the oocyte during the growth stage. But why does the oocyte not synthesize the RNAs when it needs them? A hint comes from the observation that the appearance of the small bodies occurs at the same time the nucleolus is transcriptionally inactivated, and this in turn occurs at about the same time the nucleolus starts to undergo fragmentation. It can be postulated, therefore, that these three events are related to each other: fragmentation determines a temporary inactivation of the ribosomal genes and this stop in the RNA production in turn causes a stop in the process of oocyte growth. This ‘‘danger’’ can be avoided by an early synthesis of excess rRNA and by its storage in the body with later gradual release, according to the requirements of the oocyte. The synthesis would probably restart in vitellogenesis as reported for most other vertebrate oocytes. The needs for fragmentation would be determined by the presence, in this species as in other fishes (Vlad, 1976; Raikova et al., 1979) and tetrapods (Macgregor, 1982; Motta et al., 1991) of a process of rDNA amplification. It is well known, in fact, that the formation of multiple nucleoli in the oocyte is related to the presence of extra copies of rDNA (Vincent et al., 1969). In conclusion, we hypothesize that the round body would represent a strategy to overcome the temporary stop of rRNA synthesis during the dispersion of the mass of amplified rDNA and the formation of multiple nucleoli. In other species where this body is not present, but multiple nucleoli are, other stategies may have evolved: these would represent an interesting field of investigation not only at the cytological level but also for comparative reasons. ACKNOWLEDGMENTS Research was carried out partly at the Centro Interdipartimentale di Ricerca sulle Ultrastrutture Biologiche.
204
S. FUSCO ET AL.
Figure 4. (a–f)
rRNA STORAGE IN TROUT OOCYTES
Fig. 4. Biochemical characterization of the round body: nucleic acid content. (a) Pyronin specifically stains the round body (arrow). (b) Following staining with DAPI (for AT bases), a fluorescence is observed over the round body (arrow) and the nucleoli (arrowheads). The cytoplasm, by contrast, appears unstained. (c) Following staining with distamycin (for GC bases), the round body (arrow) appears intensely fluorescent. (d) The in situ nick translation reaction reveals the presence of DNA in the oocyte nucleoplasm (N) but not in the cytoplasm (Cy) and the round body (arrow). Nucleoli (arrowheads) also appear unlabeled. (e) Treatment with the anti-DNA antibody determines a significant staining of the nuclei of the follicle cells (arrowheads) but not of the round body (arrow). Inset: detail of the nucleus of a follicle cell. Gold-particles (arrows) are concentrated over the chromatin. (f) TEM micrograph of a round body following treatment
205
with the anti-DNA antibody. Occasional gold particles (arrows) are present over the cytoplasm. (g,h) Ultrathin sections of a round body following treatment with the RNase-gold complex: gold particles are clearly concentrated over the fibrillar edges of the cords (arrows). (i) Growth stage oocytes following in situ hybridization with 18S cDNA. Labeling is specifically located over the round body (arrow) and over nucleoli (arrowheads). (j) Early growth stage oocyte following in vitro administration of tritiated uridine. Labeling is present over the follicle cells (arrows) and over the oocyte nucloplasm (N), nucleolus (arrowhead) and also cytoplasm (Cy). (k) Mid growth stage oocyte following tritiated uridine administration. Labeling is located over the nucleoplasm (N) but not over the nucleoli (arrowheads), the cytoplasm (Cy) or over the round body (arrow). a,i: 3350; b,d,k: 3800; C: 3400; e: 3650; e, inset: 350,000; f: 343,000; g: 345,000; h: 310,000; j: 3450.
206
S. FUSCO ET AL. REFERENCES
Beams HW, Kessel RG. 1973. Oocyte structure and early vitellogenesis in the trout, Salmo gairdneri. Am J Anat 136:105–122. Begovac PC, Wallace RA. 1988. Stages of oocyte development in the pipefish Sygnanthus scovelli. J Morphol 197:353–369. Bendayan M. 1981. Ultrastructural localization of nucleic acid by the use of enzyme-gold complexes. J Histochem Cytochem 29:531–541. Bendayan M. 1982. Ultrastructural localization of nucleic acid by the use of enzyme-gold complexes: influence of fixation and embedding. Biol Cell 43:153–156. Bohrmann B, Kellenberger E. 1994. Immunostaining of DNA in electron microscopy: an amplification and staining procedure for thin section as alternative to gold labeling. J Histochem Cytochem 42:635–643. Derenzini M, Farabegoli F, Trere D. 1992. Relationship between interphase AgNOR distribution and nucleolar size in cancer cells. Histochem J 24:951–956. Fusco S, Filosa S, Motta C. 1997. Previtellogenesis in Antarctic fishes: comparison with temperate species. Ital J Zool 64:209–214. Hartung M, Keeling JW, Patel C, Bobrow M, Stahl A. 1983. Nucleoli, micronucleoli, and nucleolus-like structures in human oocytes at meiotic prophase I studied by the silver-NOR technique. Cytogenet Cell Genet 35:2–8. Hernandez-Verdun D, Derenzini M, Bouteille M. 1982. The morphological relationship in electron microscopy between NOR- silver proteins and intra-nucleolar chromatin. Chromosoma 85:461–473. Karnowsky MJ. 1965. A formaldehyde-glutaraldehyde fixation of high osmolarity for use in electron microscope. J Cell Biol 27:137.
Lehmann R, Tautz D. 1994. In situ hybridization to RNA in Drosophila melanogaster: practical use in cell and molecular biology. Meth Cell Biol 44:597–598. Macgregor HC. 1982. Ways of amplifying ribosomal genes. In: Jordan EG, Kullis CA, editors. The nucleolus. Cambridge: Cambridge University Press. p 129–151. Mazzi V. 1977. Manuale di tecniche istologiche ed istochimiche. Padova: Piccin Editore. Miller CJ, Reisler E. 1995. Role of charged amino acid pairs in subdomain-1 of actin in interactions with myosin. Biochemistry 34:2694–2700. Miller OL, Beatty RR. 1969. Visualization of nucleolar genes. Scienze 164:955–957. Motta CM, Andreuccetti P, Filosa S. 1991. Ribosomal gene amplification in oocytes of the lizard P. Sicula. Mol Reprod Dev 29: 95–102. Moussa F, Oko R, Hermo L. 1994. The immunolocalization of small nuclear ribonucleoprotein particles in testicular cells during the cycle of the seminiferous epithelium of the adult rat. Cell Tissue Res 278:363–378. Raikova EV, Steinert G, Thomas CHR. 1979. Amplified ribosomal DNA in meiotic prophase oocyte nuclei of acipenser fishes. Wihelm Roux Arch 186:81–85 Vincent WS, Halsorson HO, Chen H-R, Shin D. 1969. A comparison of ribosomal gene amplification in uni- and multinucleolate oocytes. Exp Cell Res 57:250–260. Vlad M. 1976. Nucleolar DNA in oocyte of Salmo irideus (Gibbons). Cell Tiss Res 167:407–424.
