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Vitellogenic Oocytes CELL STRUCTURE AND FUNCTION 26: 693–703 (2001) © 2001 by Japan Society for Cell Biology Somatic Lamins in Germinal Vesicles of Goldfish (Carassius auratus) Vitellogenic Oocytes Akihiko Yamaguchi1 and Yoshitaka Nagahama Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan 1Present address: Laboratory of Marine Biology, Department of Animal and Marine Bioresouruce Science, Faculty of Agriculture, Kyushu Univsity, Fukuoka, Japan ABSTRACT. In fish and amphibians, B-type lamins are divided into somatic (B1, B2) and oocyte-type (B3) lamins. In this study, we purified nuclear lamins from rainbow trout erythrocytes, raised an anti-lamin mono- clonal antibody (L-200) that recognizes goldfish somatic-lamins, and isolated cDNAs encoding goldfish B-type lamins (B1 and B2) from a goldfish cell culture cDNA library. Goldfish B-type lamins are structurally similar to lamins found in other vertebrates with minor amino acid substitutions in the conserved region. Western blot anal- ysis showed that goldfish oocytes contained mainly GV-lamin B3 as well as some somatic lamins. Laser-confocal microscope observations revealed that lamin B3 was present only in GV nuclear lamina, whereas somatic lamins were present in dense fibrillar structures throughout nuclear gels of isolated GVs. Similar nuclear filamentous structures were also observed in GVs of paraffin embedded oocytes. Epitope mapping indicated that L-200 recognized a conserved region containing a short stretch of the -helix coiled-coil rod domain (Y(E/Q)(Q/E)LL). A similar motif is also present in other cytoplasmic intermediate filaments (i.e., vimentin, desmin, peripherin and GFAP). Taken together, these findings suggest that lamins or lamin-related intermediate filaments are an impor- tant component of the interior architecture of goldfish vitellogenic oocyte nuclei (GVs). Key words: actin/intermediate filament/meiosis/lamina/nuclear matrix The inner nuclear membrane of the vertebrate nucleus is Monteiro, 1998), a nuclear localization signal (NLS) intimately associated with a fibrous mesh, or lamina, com- (Loewinger and McKeon, 1988; Moir et al., 1995) and a posed of lamins that are classified into type V intermediate CaaX box (C, cystein; a, aliphatic amino acid; X, variable) filaments (IFs) (Aebi et al., 1986; McKeon et al., 1986; for which plays a role in chemical modifications such as iso- review see Stuurman et al., 1998). IFs have a central -heli- prenylation, carboxyl methylation and proteolytic trimming cal longitudinal rod-domain consisting of repeated heptads (Holtz et al., 1989; Moir et al., 1995). Lamins are classified which separate the non-helical head and tail domains thus as A- or B-type based on their primary sequence and/or sol- forming a two stranded typical coiled-coil structure (for re- ubility properties at mitosis. During interphase, lamins are view see Parry and Steinert, 1995). In contrast, lamins con- present in an insoluble form. During metaphase, however, tain 42 extra amino acids (aa) in their rod domain of six they depolymerize and become dispersed throughout the heptad repeats (Nigg, 1989; Weber et al.,1989; Mical and cytoplasm. A-type lamins depolymerize into oligomers and become completely soluble (Gerace and Blobel, 1980), *To whom correspondence should be addressed: Yoshitaka Nagahama, whereas B-type lamins remain associated with membranous Laboratory of Reproductive Biology, Department of Developmental Biolo- structures (Gerace and Blobel, 1980; Chaudhary and Cour- gy, National Institute for Basic Biology, Okazaki, 444-8585, Japan. Tel: +81–564–55–7550, Fax: +81–564–55–7556 valin, 1993). In frogs, four different lamins (LI to L III and E-mail: [email protected] LA) have been isolated and characterized throughout devel- Abbreviations: DTT, dithiothreitol; EPG, epithelioma papillosum of opment. B-type lamins are subdivided into three groups, B1, goldfish cells; GV, germinal vesicle; GFAP, glial fibrillary acidic protein; B2 and B3. Lamins B1 and B2 have been identified in IF, intermediate filament; L-200, anti-lamin monoclonal antibody; NIB, nuclei isolation buffer; NLS, nuclear localization signal, PB, phosphate somatic cells of frogs (Krohne et al., 1987; Höger et al., buffer; PBS, phosphate buffer saline; SDS-PAGE, polyacrylamide gel elec- 1990), chicks (Vorburger et al., 1989; Peter et al., 1989) and trophoresis with SDS. 693 A. Yamaguchi and Y. Nagahama mammals (Höger et al., 1988, 1990; Pollard et al., 1990). L15 medium (Sigma) containing 10% fetal calf serum at 18ºC and Lamin B3 is the only lamin specifically expressed in animal subcultured every 2–3 weeks. oocytes. Based on its primary structure, lamin B3 belongs to the B-type lamins (Stick, 1988). However, it differs from Isolation of nuclei and preparation of nuclear lamina other B-type lamins in that it becomes soluble during both Rainbow trout blood was collected by syringe with 1/10 volume of meiotic and mitotic metaphases (Benavente et al., 1985; an anti-coagulant solution [0.2 M EDTA, 100 M (p-amidino- Newport et al., 1990), although small amounts (5–7%) of phenyl) methanesulfonyl fluoride hydrochloride (p-APMSF)]. B3 and some minor B-type lamins have been reported to Erythrocytes were collected and washed in 75 mM NaCl, 25 mM remain associated with membrane fragments (Lourim and EDTA (adjusted pH 7.4 by NaOH) by centrifugation (3,000 rpm Krohne, 1993; Lourim et al., 1996). Lamins likely play for 10 min) three times. After the final spin, cells were lysed in important roles in DNA binding (Ludérus et al., 1992; nuclei isolation buffer [NIB: 10 mM Tris-HCl (pH 7.4), 10 mM 1994), changes in chromatin organization (Höger et al., NaCl, 5 mM EDTA, 1 mM spermin, 1 mM dithiothreitol (DTT), 1991; Taniura et al., 1995), nuclear structure (Furukawa and 100 M p-APMSF, 0.25 M sucrose, 0.1% Triton X-100]. Nuclei Hotta, 1993) and DNA replication (Newport et al., 1990; were then collected by centrifugation at 3,000 rpm for 5 min and Meier et al., 1991; Jenkins et al., 1993; Moir et al., 1994; washed twice in NIB at 2,000 rpm for 5 min. 2P1010 nuclei were Ellis et al., 1997; Spann et al., 1997). Consequently, the dif- isolated from 10 ml of packed erythrocytes. Goldfish erythrocyte ferences between meiotic oocyte-lamin composition and nuclei were isolated according to the methods above with the mitotic somatic lamins may have important implications for substitution of citric acid solution (pH 5.0) as the anti-coagulant. meiotic nuclear structure and function. EPG cultured cells were collected by scraping and suspended in In addition to the lamina, a second filamentous network, NIB. The suspension was passed through a 27 gauge needle to lyse i.e. the nuclear matrix, has been identified in vertebrate nu- the cells. Crude nuclei were collected by centrifugation at clei (Berezny and Coffey, 1974; Capco et al., 1982; Fey et 10,000Pg and washed with NIB several times. Isolated nuclei were al., 1986). Lamins are a structural component of the nuclear then washed with NIB and finally resuspended in NIB containing matrix as well as the peripheral lamina (Hozak et al., 1995). 50% glycerol (v/v) for storage at –80LC. Lamins appear not only with the inner nuclear membrane Somatic nuclear lamins were prepared from isolated rainbow but also with internal nucleus foci coinciding with DNA trout erythrocyte nuclei. Nuclei (2P1010) were resuspended in 20 replication in some S-phase nuclei (Moir et al., 1994). ml nuclei swelling buffer (20 mM MES, 25 mM EDTA, 1 mM Lamin foci were also identified in G1 phase nuclei (Bridger DTT, 100 M p-APMSF, 0.25 M sucrose, pH 6.5) and aliquoted et al., 1993). Intermediate filament-like structures and into eight polycarbonate tubes on ice for 15 min. 5,000 units of various other classes of filaments are present in the nuclear deoxyribonuclease II (DNase II) from beef spleen (Bioenzyme matrix (Berezney and Coffey, 1974; Capco et al., 1982; Laboratories, San Diego, USA) were added to each tube for diges- Jackson and Cook, 1988; He et al., 1991), suggesting tion of DNA. After incubation for 1 hr at room temperature, an that lamins are a likely candidate for internal nuclear equal volume of 2Pextraction buffer (20 mM Tris, 3 M NaCl, 2 matrix filaments. Internal matrix filaments were present in mM EDTA, pH 8) was added. The resulting nuclear ghosts were reconstituted sperm pronuclei in Xenopus cell free extracts pelleted at 25,000Pg for 30 min. Pellets were then suspended in 40 after removal of lamin B3 (Jenkins et al., 1993; Zhang et ml detergent buffer (20 mM MES, 0.3 M NaCl, 2 mM EDTA, 2% al., 1996). Triton X-100, 100 M p-APMSF, pH 6.0) and stirred for 30 min at In this paper, we characterized an anti-somatic lamin 4C. The nuclear lamina fraction was collected by centrifugation monoclonal antibody generated against purified rainbow (25,000Pg). Finally, the lamina were rinsed in 10 mM Tris-HCl trout erythrocyte lamins. Laser-confocal microscope analy- (pH7.4) and stored at –80LC. The insoluble lamina pellet was then sis revealed a densely packed interior filamentous nuclear used as an antigen to produce an anti-somatic lamin monoclonal structure in goldfish oocyte nuclei, whereas only the periph- antibody (L-200). Germinal vesicles (GVs) were isolated from eral nuclear lamina was recognized by an anti-lamin B3 goldfish oocytes according to the methods of Yamaguchi et al. monoclonal antibody. These results indicate that lamin-like (2001). filaments play a role in supporting nuclear structures in the interior of GVs of goldfish oocytes. Fluorescence microscope and laser scanning micro- scope observations Materials and Methods Isolated goldfish GVs were transferred into siliconized micro- Fishes and cell strains tubes, rinsed with 50 mM phosphate buffer (PB: 32.5 mM Na2HPO4, 17.5 mM NaH2PO4) and fixed in 3.7% formaldehyde/50 Rainbow trout (Oncorhynchus mykiss) were obtained from the mM PB for 30 min at room temperature.
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