Identification and molecular cloning of germinal vesicle lamin B3 ingoldfish (Carassius auratus) oocytes

Akihiko Yamaguchi1,*, Masakane Yamashita2, Michiyasu Yoshikuni1 and Yoshitaka Nagahama1

1Laboratory of Reproductive Biology, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, Japan;2Laboratory of Molecular and Cellular Interaction, Division of Biological Science, University of Graduate School of Science,

Sapporo, Japan

A bulk isolation method was developed to collect a large

number of germinal vesicles (GV) from postvitellogenic

oocytes of goldfish (Carassius auratus). Using this method,

we obtained GV lamina which are resistant to high salt and

nonionic detergent. 2D PAGE revealed that the goldfish GV

lamina contained several spots with similar molecular

masses (67 kDa) and slightly different neutral isoelectro-

focusing values (pI 5.8±6.2). After trypsin digestion and

extraction of a major spot (pI 6.1), the peptide was

subjected to RP-HPLC and sequenced. A homology search

identified this spot as a nuclear lamin. A cDNA encoding

goldfish GV lamin was isolated by RT-PCR using

degenerate primers designed from the GV lamin tryptic

peptide sequence. The goldfish GV lamin cDNA encodes a

predicted molecular mass of 67 455 Da with a pI of 5.84.

Phylogenetic analysis indicates that the amino-acid

sequence is most similar to Xenopus oocyte-specific GV

lamin B3, but differs from somatic lamins (A, B1 or B2). In

contrast to somatic lamins, neither goldfish nor Xenopus

GV lamin contain conserved phosphorylation sites for

nuclear transport, except the nuclear localization sequence.

Therefore, we conclude that the goldfish oocyte GV is

mainly comprised of GV-type lamin (the homolog of

Xenopus lamin B3).

Keywords: nuclear matrix; lamins; intermediate filament;

oocyte maturation; meiosis.

The nuclear lamina is a 10-nm thick meshwork structurethat supports an inner nuclear membrane in Xenopusoocytes [1]. Lamins are the major components of thefibrous lamina. They have molecular masses between 60and 80 kDa [2] and belong to the intermediate filamentfamily [3]. Vertebrates express a variety of lamins indifferent cell types [2]. Somatic lamins are classified intoA- and B-type by their amino-acid sequences andbiochemical characteristics. Whereas A-type is a singleisoform [4,5], two lamin B isoforms, B1 and B2, have beenidentified in birds [6,7], mammals [8±10] and amphibians(named LI and LII) [4,9]. At least one B-type lamin isconstitutively expressed [11±14]. In contrast, the expres-sion of A-type lamin is highly regulated during develop-ment such that A-type lamins are absent in early amphibian,chicken, mouse embryos [5,13±15]. Furthermore, cell typemay also affect lamin content. Reproductive cells (i.e. germcells [16]) and relatively undifferentiated cells (e.g.teratocarcinoma stem cells, [13]; T-lymphocytes [17]),contain a single lamin [18,19], but differentiated cells

[11,12] have a more complex nuclear lamina. The nuclearlamins are dynamic, changing their state of polymerizationduring the cell cycle [20]. In interphase, they are present inan insoluble form; however, during metaphase, theydepolymerize and become dispersed throughout the cyto-plasm. A-type lamins depolymerize into oligomers andbecome completely soluble. B-type remains associated withmembranous structures [21,22].

In addition to somatic lamins, several male germ cell-specific lamins including lamin LlV from Xenopus sperma-tids and sperm [16], lamin B3 and C2 of mouse and ratspermatocytes [23±25], and lamin-related peptides ofmouse [23] and rat [26,27] have been identified. However,lamins in female germ cells have not been well-character-ized. The germinal vesicle (GV) is defined as a highlyspecialized, huge nucleus of oocytes that arrests at meioticprophase I. In Xenopus, lamin Llll is a well-characterizedlamin that supports the GV inner membrane [19], cleavagenuclei [11,12] and a few, specialized cell types of adulttissues [11]. Although Llll depolymerizes to fully solubleoligomers during germinal vesicle breakdown like A-typelamin [11], its general structure is clearly B-type. Itssequence, however, matches neither the B1- nor B2-subtypesequences [28]. Consequently, it has been termed lamin B3.The primitive lamin B3 appears to be the ancestral form[29,30]. Recent studies indicated that three B-type lamins,minor somatic-lamins such as LI (B1) and LII (B2) and themajor lamin Llll (B3), were associated with the nuclearenvelope in Xenopus oocytes [31,32]. Two cDNAs of B3mRNAs differing by 12 amino acids in the C-terminalregion have also been identified [33].

In other vertebrates, it remains unclear which lamins appearin oocytes. Using a newly developed, simple method to isolate

Eur. J. Biochem. 268, 932±939 (2001) q FEBS 2001

Correspondence to Y. Nagahama, Laboratory of Reproductive Biology,

Department of Developmental Biology, National Institute for Basic

Biology, Okazaki 444-8585, Japan. Fax: 1 81 564 55 7556,

Tel.: 1 81 564 55 7550, E-mail: nagahama@nibb.ac.jp

Abbreviations: GV, germinal vesicle; GIB, GV (germinal vesicle)

isolation buffer.

*Present address: Laboratory of Marine Biology, Department of

Animal and Marine Bioresource Science, Faculty of Agriculture,

Kyushu University, Fukuoka, Japan.

(Received 22 August 2000, revised 20 November 2000, accepted

6 December 2000)

bulk GVs from fish oocytes, we investigated the biochemicalaspects of GVs from goldfish (Carassius auratus).

M A T E R I A L S A N D M E T H O D S

Animals and oocytes

Female goldfish (C. auratus) were bought from dealers andraised in the laboratory at 15 8C. Ovaries were removed anddissected into small pieces in goldfish Ringer's solution [34].

Isolation of germinal vesicles

Goldfish ovarian fragments were incubated in goldfishRinger's solution for 1 h at room temperature. Fragmentswere then transferred to Ca21 and glucose-free Ringer'ssolution containing 5 mm EGTA and incubated for anadditional 2 h. Follicle-enclosed oocytes were isolated bygentle pipetting with plastic bellow pipette (2±3 mm in thediameter of tip width) every 30 min. Partially naked(follicle-free) oocytes were obtained during this incubation.Isolated oocytes were poured into 50 mL conical plastictube containing ice-cold GV isolation buffer [GIB, 10 mmTris/HCl pH 7.4, 33 mm NaCl, 7 mm KCl, 1 mm spermine,0.2% Triton X-100, 50 mm (p-amidinophenyl)methane-sulfonyl fluoride hydrochloride] and agitated by gentlepipetting. Small oocytes and connective tissues wereremoved by decantation, leaving large, fully grown oocytes.Packed, full-grown oocytes (25±40 mL) were usuallyincubated in 200 mL ice-cold-GIB on ice without shaking.After 1 h, the GIB was decanted and replaced. The oocyteswere incubated for an additional hour on ice. GVs werereleased from the oocytes during incubation (see the figuresof the results). Most debris was removed by filtrationthrough a nylon mesh (with a hole of 0.5 mm). IsolatedGVs were transferred to 15-mL plastic tubes on ice. GVswere collected at 1 g for 1 h. As some oocytes containingGVs remained after the initial treatment, further incuba-tions with fresh GIB were carried out to collect these GVs.Bulk, isolated GVs were immediately used to prepare GVlamina. When using less than 50 GVs, they were manuallyisolated from immature oocytes in Ringer's solution.

Preparation of lamin enriched residual GV membranes(GV lamina)

Isolated GVs (5 � 103) were suspended in 500 mL ofnucleic acid digestion buffer [50 mm Tris/HCl pH 7.5,100 mm NaCl, 8 mm MgSO4, 50 mm (p-amidinophenyl)methanesulfonyl fluoride hydrochloride]. One hundredunits of DNase I (Takara) and pancreatic RNase A(Boehringer) were added for digestion of nucleic acids.GVs were then incubated for 1 h at room temperature.After a brief spin, GVs were resuspended in 1 mL of highsalt extraction buffer [10 mm Tris/HCl pH 7.4, 1 m KCl,1% Triton X-100, 0.1 mm EDTA, 1 mm dithiothreitol,50 mm (p-amidinophenyl) methanesulfonyl fluoride hydro-chloride] and rotated for 1 h to overnight at 4 8C. Afterbrief centrifugation, pellets were resuspended in thesame buffer and incubated further. GV lamina werecollected at 10 000 g for 10 min and rinsed with 10 mmTris/HCl (pH 7.4) solution several times and storedat 280 8C.

Sections of isolated GVs and residual GV envelopes

Isolated GVs or GV lamina were precipitated by low-speedcentrifugation. The sediments were fixed with 1% osmic-acid and embedded in epoxy resins. One-micrometersections were prepared and stained with methylene-blue.Photographs were taken using Biophot (Nikon).

Extraction of proteins

Goldfish GV proteins were extracted by homogenizingisolated GVs in lysis buffer (9 m urea, 2% Triton X-100,2% 2-mercaptoethanol, 0.8% ampholine 3-10) with amicropestle and centrifugating at 10 000 g for 30 min.The supernatant containing the GV proteins was collected.GV lamins were simultaneously extracted from theprepared GV lamina by the same method. Soluble extractwas centrifuged twice at 100 000 g for 1 h. The supernatantwas concentrated and excess salts were removed bymicroconcentrators (Centricon-10, Amicon). Solid ureaand other reagents were added to the final concentrationof lysis buffer.

Electrophoresis

For SDS/PAGE, the extracts or immunoprecipitated pro-tein A-conjugated beads were mixed with an equal volumeof 2 � SDS sample buffer and boiled for 1 min. Proteinswere separated by SDS/PAGE (7.5% polyacrylamide [35]).2D gel electrophoresis (IEF/SDS/PAGE) was carried oututilizing the Multiphore 2 two-dimensional gel system(Pharmacia). For the first-dimension, an immobilized IEFgel (Drystrip pH 4±7) was used. GV lamina equivalent to2 � 103 GV were dissolved in 25 mL of lysis buffer anddiluted with an equal volume of sampling buffer (8 m urea,0.5% Triton X-100, 2% 2-mercaptoethanol, 0.8% ampho-line pH 3±10) and loaded on the gel directly. An excel-gelSDS gradient (8±18%) was used in the second dimension.Proteins were stained with Coomassie-brilliant blue (1D) orwith silver-staining (2D).

In gel digestion, peptide purification and sequencing

Lamina proteins from 1 � 105 GV were separated on fivepieces of 2D polyacrylamide gels. Spots were stained with0.2% Coomassie brilliant blue R-250 in 40% methanol, 1%acetic acid solution. Spots (67 kDa, pI 6.1) were excised,pooled and subjected to in gel digestion. Five gel spots werepooled, washed with washing buffer (50% acetonitrile,0.2 m ammonium bicarbonate), dried, re-swollen andincubated with 1 mg sequence-grade modified trypsin(Promega) in cleavage buffer (0.2 m ammonium bicarbo-nate, 2 m urea) for 17 h at 37 8C. The reaction was stoppedby adding trifluoroacetic acid to a final concentration of1%. After centrifugation, the supernatant was collected.Peptides were eluted from gels by stirring twice with200 mL elution buffer (60% acetonitrile, 0.1% trifluoroa-cetic acid). The eluates were concentrated to one-third theiroriginal volume by speed vacuum. Supernatants and eluatefrom the gels were mixed and applied to RP-HPLC (Smartsystem, Pharmacia). Peptides were separated using a mRPCSC2.1/10 column (Pharmacia) with a gradient of water/0.065% trifluoroacetic acid to 45% acetinitorile/0.05%

q FEBS 2001 Characterization of goldfish oocyte lamins (Eur. J. Biochem. 268) 933

trifluoroacetic acid at a flow rate of 100 mL´min21.Sequence analysis of RP-HPLC separated peptides wascarried out by automatic sequencing (492, AppliedBiosystems).

Screening of cDNA encoding goldfish GV lamin and DNAsequencing

A goldfish GV lamin cDNA was isolated using RT-PCR.First strand cDNA was synthesized from 100 ng goldfishovary polyA RNA by 200 U Superscript II (BRL) in12 mL total volume using 3 0-RACE oligo dT/adaptor/primer [1 mg, 37-mers; 5 0-GGCCACGCGTCGACTAG-TACT(T)16-3 0]. PCR was carried out in 50 mL total volumecontaining 1 mL of 1st strand cDNA, 50 pmol each forwardand reverse primers and 2.5 U Takara LA Taq polymerase.Cycling conditions were as follows: 30 cycles of 1 min at95 8C, 1 min at 50 8C, and 2 min at 72 8C. Primers weredesigned from amino-acid sequences of tryptic peptidesfractionated with HPLC. At first, a combination of forwardprimer A, 5 0-GA(TC)GCNGA(AG)AA(TC)CA(AG)CTN-CA(AG)AC-3 0 designed from fraction 37, and reverseprimer C, 5 0-AC(TC)TCNCC(AG)AANGGNCCCCA-3 0from fraction 33 were used to amplify GV lamin DNA.As the PCR products were only faintly visible, a secondPCR using the forward nested primer B, 5 0-CA(AG)-GA(AG)CA(AG)CTNGA(TC)TT(TC)CA(AG)AA-3 0 fromfraction 37 and reverse primer C was carried out. A 977-bpfragment was amplified, ligated to the TA cloning vector,PCR2 (Invitrogen) and sequenced. cDNA probes wereprepared with a random labeling kit (NEN) and hybridizedto a ZAP cDNA library derived from ovarian polyA RNA.

Two full-length positive clones encoding lamin B3 wereisolated from 2 � 105 plaques. Both encoded the samelamin B3.

Phylogenetic analysis

Lamin overall protein sequences were aligned usingclustalw, a multiple sequence alignment program(http://www.ddbj.nig.ac.jp/htmls/E-mail:/homology-j.html).A phylogenetic tree of amino-acid sequences was con-structed by the neighbor joining method [36] using thephylip 3.573c program written by J. Felsenstein (Uni-versity of Washington, Seattle, WA, USA). The evolu-tionary distance between two aligned amino-acid sequenceswas measured by the Dayhoff PAM matrix. The robustnessof the phylogenetic hypothesis was tested by bootstrapping[37]. In this study, all boot strap analyses of amino acidsinvolved 100 replications of the data.

R E S U L T S

Bulk GV isolation from goldfish oocytes

As fish GV lamins have not yet been characterized, wedeveloped a simple method to isolate enough GVs tobiochemically characterize lamins. Gentle incubation offollicle-free oocytes in hypotonic medium (GIB) containingspermine and nonionic detergent enabled the isolation ofintact GVs. For a high yield of GVs, oocytes must beincubated in fresh Ca21-free Ringer's solution to denudethem by pipetting before being transferred to GIB. Withoutremoval of the follicle, oocytes could not swell in thehypotonic medium used in the next step. Incubation in GIBcauses swelling that appears to result in cracking at thededuced micropyle position. GVs were extruded throughthis crack (Fig. 1A). Excessive incubation of oocytes in

Fig. 1. Mass isolation of GVs from goldfish oocytes (A, B). and

sections of an isolated GV (C) and GV lamina (D). (A) A GV

extruding from an oocyte through the pore (arrowhead). (B) Isolated

GVs after rinse. (C) The nuclear gel is surrounded by the nuclear

membrane. (D) Residual nuclear membranes are layered after nuclear

contents are removed by salt and detergent treatments. Bar � 500 mm

(A, B), 100 mm (C, D.)

Fig. 2. SDS/PAGE analysis of GV lamina. Extracts from total GV-

proteins (from 100 GV, lane 1), soluble nucleoplasmic fractions

prepared after high salt treatment (from 100 GV, lane 2) and insoluble

lamina fractions (from 400 GV, lane 3) were separated on 7.5% SDS/

PAGE and stained in Coomassie-brilliant blue. The left lane indicates

molecular mass markers stained with Coomassie. The arrowhead

indicates the position of 67-kDa protein (GV lamin).

934 A. Yamaguchi et al. (Eur. J. Biochem. 268) q FEBS 2001

GIB results in contamination by yolk platelets. Thecomposition of GIB is also important for GV isolation. ANP-40 concentration of 0.2% is suitable to keep the nuclearmembrane intact and disperse the contaminating yolkplatelets in GIB. The hypotonic isolation buffer causedthe nuclear envelope of most GVs to separate from thenuclear gel containing nuclear materials (Fig. 1B). Avisible nuclear gel inside the intact nuclear membranewas observed (Fig. 1C). The number and localization ofnucleoli inside the GV were dependent upon the stage ofthe oocytes prior to GV isolation.

A 67-kDa peptide is a major component of the GV lamina

When bulk isolated GVs were vigorously vortexed withhigh salt buffer, the internal structure of the GV (nucleargel) breaks down leaving the GV lamina as an insolublepellet. The sections of this insoluble pellet seem to be thelayers of residual GV-membranes (Fig. 1D). The proteincomposition of the GV lamina was analyzed by 1D SDS/PAGE. Whereas most GV proteins were extracted with thesalt/detergent soluble nucleoplasmic fraction, a 67-kDapeptide (p67) remained as a major component of GVlamina (Fig. 2). 2D (IEF/SDS) PAGE analysis (Fig. 3)showed that GV lamina has several spots with similarmolecular masses (67 kDa) but slightly different isoelectro-focusing points of lamin B3 (pI 5.8±6.2) including twomajor spots (pI 5.9 as spot a, 6.1 as spot b). Therefore, theseresults strongly suggest that goldfish GV lamin is a 67-kDalamin with a pI of 5.8±6.2.

Amino-acid sequencing and cDNA cloning of goldfish GVlamins

To determine amino-acid sequences of goldfish lamins,insoluble lamina fractions from 1 � 105 GV were separatedon five pieces of 2D SDS/PAGE gels, subjected to trypticdigestion, RP-HPLC and automatic amino-acid sequencing.Two peak fractions were sequenced. Fraction 33 containedthe 12 amino-acid tryptic peptide THETWGPFGEVR thatshowed no homology with any known proteins. Fraction 37contained the 18 amino-acid peptide VDAENQLQTL-QEQLDFQK that showed high homology with lamins. A977-bp DNA fragment was amplified by PCR using primers

Fig. 3. 2D polypeptide patterns of goldfish GV lamina. GV lamina

proteins (2 � 103) were separated on 2D PAGE and silver stained.

Molecular masses (Mr) of reference proteins are in kDa. a, pI 5.9; b, pI 6.1.

Fig. 4. Unrooted phylogenetic tree of lamin proteins was constructed with the neighbor joining method. The numbers indicate bootstrap values

from 100 replicates. Lines indicate genetic distance. Lamin protein sequences from the current release of SwissProt and GenBank databases: the

snail lamin-like intermediate filament, S12277; the Caenorhabditis elegans lamin, CAA52188; the Drosophila melanogaster lamin Dmo, P08928;

the Drosophila melanogaster lamin C, Q03427; the sea urchin B-type lamin, AAB34118; the Xenopus lamin A; P11048; the Xenopus lamin B1,

P09010; the Xenopus lamin B2, P21910; the Xenopus lamin B3, P10999; the chicken lamin A, P13648; the chicken lamin B1, P14731; the chicken

lamin B2, P14732; the mouse lamin A, P48678; the mouse lamin B1, P14733; the mouse lamin B2, P21619; the zebrafish (Danio rerio) lamin B1,

AJ250201; the zebrafish lamin B2, AJ005936; the goldfish lamin B2, AB034198; the goldfish lamin B3, AB034l97.

q FEBS 2001 Characterization of goldfish oocyte lamins (Eur. J. Biochem. 268) 935

designed against the predicted nucleotide sequence of thesepeptides. Sequence analysis revealed that this fragmentencodes the nuclear lamin including the entire coil 2domain, part of coil 1b, and most of the tail region.However, it was found that the sequence of this fragmentdiffered from that of goldfish lamin B1 (DDBJ accessionnumber, AB034199) and B2 (AB034198) isolated fromepithelioma papillosum of goldfish culture cells. We thencloned the full-length goldfish GV lamin cDNA using thisfragment as a probe. The goldfish GV lamin codes for 589amino acids with a predicted molecular mass of 67 455 Daand a pI of 5.84. It contains a putative nuclear localizationsequence as well as the rod domain and CaaX box at theC-terminal end. It does not have regions characteristic of

A-type lamins such as a longer tail domain with histidinerepeats.

Phylogenetic analysis indicates that vertebrate laminsare classified into three major branches, A, B1 and B2.Furthermore, goldfish and Xenopus lamin B3 are groupedtogether but differ significantly from other vertebratelamins (A, B1 and B2) in their primary structure (Fig. 4).The overall amino-acid sequence of goldfish GV lamin wasmost similar to Xenopus lamin B3 (50% identity, Fig. 5).Sequence identity between goldfish and Xenopus lamin B3of each domain is 42% (head), 70% (coil 1a), 56% (coil1b), 58% (coil 2), and 34% (tail). Two cdc2 phos-phorylation sites, which are known to be necessary forinduction of lamina disassembly, are conserved. The

Fig. 5. Alignment of the deduced

amino-acid sequence of goldfish lamin B3,

Xenopus lamin B3, and zebrafish lamin

B3 (EST-clone, AW153707). Coil 1a, 1b

and 2 are indicated. The nuclear localization

sequence (NLS) and CaaX box sequences are

underlined. Boxes indicate the matching

amino-acid sequences of goldfish, Xenopus

and zebrafish lamin B3. The degree of

identity between the two fish lamin proteins

in the head domain, coil 1a and coil 1b is

much higher (80%) than between the goldfish

and the Xenopus protein.

Fig. 6. Phosphorylation sites homologous

between B-type lamins. Lamin amino-acid

sequences were aligned in the region

between the conserved N-terminal p34cdc2

phosphorylation sites, the beginning of coil

1a containing protein kinase A (PKA) site

and the beginning of tail containing p34cdc2

and protein kinase C (PKC) phosphorylation

sites, respectively. Boxes indicate conserved

phosphorylation sites between B-type

lamins. B3-type lamins have conserved

phosphorylation sites except PKC sites

beside nuclear localization sequence. GF,

goldfish; Xe, Xenopus; Ch, chicken.

936 A. Yamaguchi et al. (Eur. J. Biochem. 268) q FEBS 2001

protein kinase C sites, except the nuclear localizationsequence, which inhibit in vitro transport of chick laminB2 were absent. These protein kinase C sites are alsoabsent in Xenopus lamin B3, but conserved among othersomatic lamins (Fig. 6). Based on these results, weconclude that goldfish GV lamin is homologous to Xenopuslamin B3.

D I S C U S S I O N

Characteristics of GV lamins (B3)

We identified a novel lamin from female germ cells ofgoldfish. 2D PAGE analysis showed that goldfish GV laminis 67 kDa with a neutral isoelectric point [2,5,6,8] betweenthe acidic B-type and basic A-type somatic lamins. Theisoelectric point therefore did not suggest that GV laminwas either A- or B-type. Other female GV lamins, i.e.Xenopus lamin B3 and surf clam L67, also have neutralisoelectric points of 6.5 and 6.3, respectively [11,18].Consequently, neutral isoelectric values appear to be acharacteristic of lamins expressed in oocytes. Maul et al.[18] strongly suggested that clam lamins were cross-linked by disulfide bonds; however, we were unableto determine any involvement of disulfide bonds ingoldfish GV lamin in its unreduced state by iodoacetamidetreatment. The spot with a pI of 5.9 contained the sametryptic peptides as the pI 6.1 spot (data not shown).Therefore, we think that these spots are derived fromthe same peptide and subjected to modification such asphosphorylation.

Protein sequence analysis indicates that B3-type lamindiffers significantly from other vertebrate A- and B-typelamins. Mouse, chicken and Xenopus A- and B-type lamins(B1 and B2) are grouped closely. In contrast, goldfishand Xenopus B3 are much further apart genetically.Despite this genetic separation, the biochemical charac-teristics of B3 are conserved, i.e. they become fullysoluble oligomers during oocyte maturation withoutmembrane binding similar to A-type lamins. This con-served biochemical feature suggests that lamin B3 isimportant for oocyte growth, oocyte maturation and earlyembryogenesis.

Recently, unusual lamins have been reported in inverte-brates. Celam-1 from Caenorhabditis elegans is a B-typelamin with a truncated coil 2 region, a shorter tail domain,and no lamin-specific cdc2-kinase motif [38]. pG-IF(intermediate filament) from Drosophila melanogasterlacks a CaaX box and has a neutral pI value [39].

Our unrooted tree indicated that vertebrate lamins differsignificantly from invertebrates in their primary structure.Recent analysis of the gene structure of lamins andinvertebrate intermediate filament proteins, however,found high overall similarity of the intron position encodingXenopus lamin B3 and snail (Helix aspersa) intermediatefilament protein [29,33]. Goldfish lamin B3 gene structureanalysis should help clarify the evolution of the lamin/intermediate filament.

Conserved phosphorylation sites of lamin B3

Lamins are phosphorylated by multiple kinases and containseveral conserved phosphorylation sites [40]. Although

p34cdc2 kinase is a mitotic kinase which induces lamindisassembly [41±43], it remains unclear whether phos-phorylation by p34cdc2 kinase induces lamina disassemblyduring GV breakdown. Conserved phosphorylation sitessuggests that a p34cdc2 phosphorylation site is also presentin GV lamins of fish and frogs (Fig. 6). A protein kinase Asite, which inhibits lamin polymerization, is also conservedin all species [44], suggesting that p34cdc2 and proteinkinase A are candidate lamin kinases for germinal vesiclebreakdown as well as somatic cell division. One additionalfunction of the protein kinase C phosphorylation site is toregulate lamin uptake into the nucleus [45]. The absenceof a protein kinase C site in GV lamins suggests thatlamin transport may be different between somatic nucleiand GVs.

Bulk GV isolation

We developed a bulk GV isolation method to purify lamins.This method utilizes no enzymes and has the advantage ofavoiding centrifugation which typically results in loss offragile GVs. Our isolation procedure can be used toelucidate the structures and functions of fish GVs fromsmall previtellogenic oocytes as well as large vitellogenicoocytes. The morphology of isolated GVs was found to besimilar to manually isolated GVs in Ringer's solution (datanot shown). Nevertheless, the nuclear membrane separatesfrom the gelatinous balls during the isolation process inboth isolation methods. This may be caused by an increasein the volume of nuclei due to the use of hypotonicmedia. Further studies are necessary to determine variousnuclear activities of GVs (e.g. transcription, recombination,chromatin assembly, etc.; [46±48]) isolated under theseconditions.

A C K N O W L E D G E M E N T S

We are grateful to Ms Y. Makino (the Center for Analytical

Instruments, NIBB) for carrying out the amino-acid sequencing, and

Dr C. E. Morrey for reading the manuscript. This research has been

supported in part by Grants-in-Aids from the Japan Society for the

Promotion of Science (JSPS-RFTF 96L00401) and Scientific Research

(07283103, 08454266, 10440247) from the Ministry of Education,

Science, Culture, and Sports, Japan.

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