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 organ- elle 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.  2000 Wiley-Liss, Inc. Key Words: rRNA; storage; oocytes; trout 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 Oncorhyn- chus mykiss, in order to clarify their function during oocyte growth. MATERIALS AND METHODS Light Microscopy and Immunocytochemistry Ovaries dissected from animals obtained from com- mercial 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 stan- dard procedures. Slides were then stained with haema- toxylin-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 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 assis- tance of Dr. Rosaria Scudiero at the Istituto di Bio- chimica delle Proteine ed Enzimologia, CNR, Naples. Electron Microscopy and Immunocytochemistry Samples for morphological observations were fixed according to Karnowsky (1965), dehydrated in ethanol, and embedded in Epon 812 following the manufactur- er’s instructions. Ultrathin sections were double- stained with uranyl acetate and lead citrate and ob- served under a model 301 Phillips electron microscope. Samples for immunocytochemical revelation of the DNA were prepared according to Bohrmann and Kellen- berger (1994) using a mouse primary antibody (Boehringer, 1:8 dilution) and a secondary antibody labeled with colloidal gold (10 nm, Sigma, 1:20 dilu- tion). 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. 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 Ani- mali.’’ *Correspondence to: Chiara Motta, Dip. Biologia Evolutiva e Com- parata, via Mezzocannone 8, 80134 Napoli, Italy. E-mail: filosa@dgbm.unina.it Received 25 October 1999; Accepted 27 December 1999 MOLECULAR REPRODUCTION AND DEVELOPMENT 56:198–206 (2000)  2000 WILEY-LISS, INC.