REGULAR ARTICLE

Balbiani cytoplasm in oocytes of a primitive fish,the sturgeon Acipenser gueldenstaedtii, and its potentialhomology to the Balbiani body (mitochondrial cloud)of Xenopus laevis oocytes

Monika Zelazowska & Wincenty Kilarski &Szczepan M. Bilinski & Daniel D. Podder &

Malgorzata Kloc

Received: 5 December 2006 /Accepted: 19 February 2007 / Published online: 16 March 2007# Springer-Verlag 2007

Abstract The oocytes of many organisms, including frogsand fish, contain a distinct cytoplasmic organelle called theBalbiani body. Because of the scarcity of publishedinformation and the tremendous variability in the appear-ance, ultrastructure, and composition of Balbiani bodiesbetween species, the function of the Balbiani body and itsinter-species homology remain a mystery. In Xenopuslaevis, the Balbiani body is known to play a role intransporting germ cell determinants and localized RNAs tothe oocyte vegetal cortex. In fish, however, the molecularcomposition of the Balbiani body has not been studied todate, and its function remains completely unknown. Wehave studied the ultrastructure and molecular compositionof previtellogenic oocytes of the sturgeon, Acipensergueldenstaedtii, by using electron microscopy, in situhybridization, and immunostaining. We have found that

sturgeon oocytes contain two distinct zones of cytoplasm:homogeneous (organelle-free) and granular (organelle-rich).We have also found that the granular ooplasm, which weterm the Balbiani cytoplasm, shares important homologies,in both ultrastructure and molecular composition, withXenopus Balbiani bodies.

Keywords Oogenesis . RNA localization . Balbiani body .

Sturgeon, Acipenser gueldenstaedtii (Chondrostei) .

Xenopus laevis (Anura)

Introduction

The oocytes of many invertebrate and vertebrate speciescontain a highly distinct, peculiar cytoplasmic structurecalled, after the name of its discoverer, the Balbiani body(Kloc et al. 2004a). Because of the scarcity of publishedinformation and the tremendous variability in the appear-ance, ultrastructure, and composition of Balbiani bodiesbetween species, the function of this body and itsfunctional, structural, or molecular inter-species homologyremain a mystery (Kloc et al. 2004b). So far, the onlyspecies in which the ultrastructure, molecular composition,and function of the Balbiani body have been studied indetail is the frog Xenopus laevis. In Xenopus, the Balbianibody plays the role of a vehicle transporting germ celldeterminants (germ plasm and its germinal granules) andlocalized RNA to the oocyte vegetal cortex (Forristall et al.1995; Kloc and Etkin 1995). The Xenopus Balbiani body,also called the mitochondrial cloud, forms during earlyoogenesis. In oogonia, the Balbiani body is visible in the

Cell Tissue Res (2007) 329:137–145DOI 10.1007/s00441-007-0403-9

This work was supported by funds from the research grant BW/IZ/2005 to M.Z.

M. Zelazowska : S. M. BilinskiDepartment of Systematic Zoology, Institute of Zoology,Jagiellonian University,Krakow, Poland

W. KilarskiDepartment of Cytology and Histology, Institute of Zoology,Jagiellonian University,Krakow, Poland

D. D. Podder :M. Kloc (*)Department of Biochemistry and Molecular Biology,M. D. Anderson Cancer Center, University of Texas,1515 Holcombe Boulevard,Houston, TX 77030, USAe-mail: mkloc@mdanderson.org

vicinity of the nuclei as an electron-dense nuage (theprecursor of germinal granules) surrounded by numerousmitochondria (Kloc et al. 2004a). In previtellogenic stage Ioocytes, the Balbiani body is a spherical structure thatcontains hundreds of thousands of mitochondria, severalhundred germinal granules (collectively called the germplasm), localized RNAs (such as Xcat2, Xlsirts, and Xpat),RNA-binding proteins, endoplasmic reticulum, and Golgicomplexes (Bilinski et al. 2004; Eddy 1975; Heasman et al.1984; Kloc et al. 2002, 2004a,b; Song et al. 2007). Duringprevitellogenesis and vitellogenesis, the Balbiani bodyexpands between the nucleus and the vegetal pole of theoocyte, delivering mitochondria, germinal granules, andlocalized RNAs to the oocyte vegetal cortex, and subse-quently fragments and disperses in the vegetal cytoplasm(Heasman et al. 1984; Kloc and Etkin 1995, 1998; Klocet al. 1998; Wilk et al. 2005).

In most teleost fish, the Balbiani body is, as in Xenopus,a prominent feature of previtellogenic oocytes (Azevedo1984; Beams and Kessel 1973; Begovac and Wallace 1988;Kobayashi and Iwamatsu 2000; Selman and Wallace 1989).Although the morphology of the Balbiani body is highlyvariable, even between closely related fish species, itusually contains a nuage, mitochondria, multivesicularbodies, endoplasmic reticulum, and Golgi complexes. Inthe majority of teleosts studied, the Balbiani body appears,as in Xenopus, during early oogenesis in close proximity tothe oocyte nucleus; during previtellogenesis, it migrates tothe oocyte periphery and disperses into small fragments.This process has been followed in living oocytes of the gulfpipefish, Syngnathus scovelli (Syngnathiformes; Begovacand Wallace 1988). However, the molecular composition offish Balbiani bodies has not been studied to date, and theirfunction remains a complete mystery. Moreover, to compli-cate the matter, the Balbiani body of some fish species, such asOryzias latipes, coexists with an irregularly shaped zone ofbasophilic cytoplasm, which surrounds the germinal vesicle(Kobayashi and Iwamatsu 2000), whereas in other species,such as Fundulus heteroclitus, only this zone of basophiliccytoplasm is present (Selman and Wallace 1986). Thispeculiar zone of cytoplasm and its structural or functionalrelationship to the Balbiani body have never been studied.

The process of oogenesis in evolutionarily old fishspecies, such as sharks, sturgeons, and lungfish, has notbeen thoroughly studied. Nevertheless, oocytes of thelungfish, Protopterus annectus (Dipnoi), have been shownnot to contain Balbiani bodies (Johnson et al. 2003),whereas oocytes of sturgeons possess a prominent Balbianibody composed of lipid droplets (Zelazowska et al. 2006).In the current study, we have examined the ultrastructureand molecular composition of previtellogenic oocytes of thesturgeon, Acipenser gueldenstaedtii, by using electronmicroscopy, in situ hybridization, and immunostaining.

We have found that, in A. gueldenstaedtii, the oocytecytoplasm (ooplasm) contains two distinct zones: a homo-geneous (organelle-free) and a granular (organelle-rich)zone. We have also found that the granular ooplasm sharesimportant homologies, in both ultrastructure and molecularcomposition, with the Xenopus Balbiani body.

Materials and methods

Electron microscopy

Specimens of A. gueldenstaedtii were bred in the Instituteof Inland Fisheries (Olsztyn-Kortowo, Poland) and in theInstitute of Ichthyology and Fisheries (Agricultural Acad-emy, Krakow, Poland) in artificial ponds. Small pieces ofovaries were removed surgically from mature females(∼1 m long), immediately immersed in ice-cold 2.5%glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), fixedfor 5–6 h, rinsed, and postfixed in 1% osmium tetroxide inthe same buffer. After dehydration in a series of ethanol andacetone washes, the tissue samples were embedded in Epon812 (Agar, Stansted, UK). Semi-thin sections (0.7 μm)were stained with 1% methylene blue in 1% borax andexamined under a Leica DMR microscope (Wetzlar,Germany). The contrast of some sections was enhancedwith Nomarski differential interference contrast (DIC).Ultrathin sections (90 nm) were contrasted with uranylacetate and lead citrate and examined under a JEOL SX orZeiss EM10 electron microscope, at 80 kV.

In situ hybridization and immunostaining

Small fragments of ovaries at various stages were fixed inMEMFA (0.1 MMOPPS, 2 mM EGTA, 1 mMMgSO4, 3.7%formaldehyde) for 1–2 h, transferred to 100% methanol, andstored at −20°C. After being cleared in HistoClear (NationalDiagnostic), samples were embedded in Paraplast andsectioned at 10 μm. Sections were deparaffinated inHistoClear, rehydrated through a series of ethanol and waterwashes, and used for immunostaining or in situ hybridization.

For immunostaining, rehydrated samples were blocked incasein blocking solution (Biorad) for 1 h and then incubatedfor 1 h with the anti-Vera (gift from B. Schnapp, OregonHealth Sciences University, Portland, OR) and anti-Fatvgrabbit polyclonal antibodies diluted 1:500 with the blockingbuffer. After several 10-min washes with 0.05% Tween inphosphate-buffered saline (PBS-0.05% Tween), samples wereincubated for 1 h with an anti-rabbit antibody (Calbiochem)conjugated with alkaline phosphatase (1:500 dilution inblocking buffer). Subsequently, following several 10-minwashes with PBS-0.05% Tween, samples were stained withalkaline phosphatase substrate (NBT/BCIP tablets [nitroblue

138 Cell Tissue Res (2007) 329:137–145

tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate]; Roche).After post-fixation in 4% formalin in PBS-0.05% Tween,samples were analyzed with a Leica DMR microscope. Incontrol experiments, sections were treated as described above,but the primary antibody was omitted.

In situ hybridization with digoxigenin-labeled antisenseRNA probes for Xcat2, Xpat, Xdazl, and nanos (Forristallet al. 1995; Houston and King 2000; Hudson and Woodland1998; Nüsslein-Volhard et al. 1987) and with sense controlprobes was performed as previously described (Bradley et al.2001; Kloc and Etkin 1999). To find even the shortestregions of possible homology between Xenopus and Dro-sophila probes and target sturgeon RNAs, the probes werehydrolyzed for 50 min to fragments of 20–50 bp, asdescribed previously (Kloc and Etkin 1999; Bradley et al.2001). After post-hybridization washes, samples were incu-bated for 1 h with the anti-digoxigenin antibody (Roche)conjugated with alkaline phosphatase (1:5000 dilution inblocking buffer), stained with alkaline phosphatase substrateNBT/BCIP tablets (Roche), post-fixed in MEMFA, andexamined under a Leica DMR microscope.

Results

Gross morphology of oocytes

We found that oocytes of A. gueldenstaedtii were sur-rounded by two layers of somatic (mesodermal) cells: anexternal layer of thecal cells and an internal layer composedof follicular cells (not shown). The follicular cells werenoticeably flattened, and their apical surfaces remained indirect contact with the oocyte plasma membrane(oolemma). The basal surfaces of follicular cells werecovered by a thick basal lamina (Fig. 1a). To facilitatefurther description, we classified oocytes into five devel-opmental categories: early previtellogenic (stage 1) oocytes,mid-previtellogenic (stage 2) oocytes, late-previtellogenic(stage 3) oocytes, vitellogenic (stage 4) oocytes, andpostvitellogenic (i.e., covered with zona radiata, stage 5)oocytes. The above classification was based on the oocytediameter, number of lipid droplets, amount of yolk, andcyto-architecture of the ooplasm (for details, see below).Here, we describe the results of ultrastructural andmolecular analysis of stage 1–3 previtellogenic oocytes.Forthcoming publications will be devoted to the ultrastruc-ture and function of a lipid body and the formation of theegg envelope.

Oocyte ultrastructure

Stage 1 oocytes were spherical and measured ∼50 μm indiameter (Fig. 1a). At this stage of development, the oocyte

nucleus (germinal vesicle) had a slightly undulated nuclearenvelope (Fig. 1c) perforated by numerous pore complexes.As in Xenopus, the germinal vesicle contained severaldense nucleoli, most likely formed as a result of ribosomalDNA (rDNA) amplification, immersed in the transparentkaryoplasm (Fig. 1a,c). The nucleoli were preferentiallylocated near the nuclear envelope (Fig. 1a,c). The oocytecytoplasm (ooplasm) was differentiated into two morpho-logically distinct zones, which we termed the homogeneous(organelle free) ooplasm and the granular (organelle-rich)ooplasm (Fig. 1a). The granular ooplasm resided in thecenter of the cell and completely surrounded the germinalvesicle, and the homogeneous ooplasm was located at theoocyte periphery. The granular ooplasm formed irregularradially arranged strands that penetrated the homogeneousooplasm, partitioning it into different size islands (Fig. 1a).Granular ooplasm contained numerous easily recognizableorganelles (see below) and lipid droplets. The lipid dropletsoften formed a single large accumulation located in thevicinity of the germinal vesicle (Figs. 1a, 2b), whichsubsequently will be referred to as the lipid body. In aprevious report, this accumulation of lipid droplets wastentatively and, in our opinion, incorrectly (see below)termed the Balbiani body (Zelazowska et al. 2006). Ourelectron-microscopic analysis showed that the homoge-neous ooplasm contained only ribosomes, whereas thegranular ooplasm contained mitochondria, elements ofrough endoplasmic reticulum (RER), Golgi complexes,ribosomes, accumulations of nuage material, and nuage/mitochondria complexes (Fig. 1c,d). Accumulations ofnuage material and the nuage/mitochondria complexeswere found almost exclusively around the germinal vesicle(Fig. 1c) and were not present within the homogeneousooplasm or in the vicinity of the lipid body. Our analysis ofhundreds of electron micrographs (EMs) indicated thatsmall initial nuage accumulations (Fig. 1c; arrowheads)coalesced into larger spherical aggregates (Fig. 1c, arrows)that were gradually surrounded by mitochondria and RERvesicles. These led to the formation of characteristic rosette-like complexes in which mitochondria and RER vesiclessurrounded centrally located nuage aggregates (Fig. 1f,g).The subsequent stages of the formation of these complexesare shown in Fig. 1c,d.

In stage 2 oocytes (60–100 μm in diameter), both typesof ooplasm (homogeneous and granular) were still clearlyrecognizable; however, their spatial arrangement wassubstantially altered. The homogeneous ooplasm formedsmall isolated islands distributed throughout the oocyte andcontained ribosomes only (Fig. 1f). These islands wereseparated by irregular strands of granular ooplasm(Fig. 1b). The strands contained numerous cisternae ofRER, mitochondria, ribosomes, Golgi complexes, andnuage/mitochondria complexes (Fig. 1f,g). Analysis of

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semi-thin sections by DIC optics in order to obtain moredetailed images of organelle distribution revealed that RER,visible on EMs as individual short cisternae (Fig. 1e,f), wasactually an elaborate anastomosing network of RER(Fig. 1b). In addition, we found that mitochondria werelocated at the peripheries of the strands often extending intothe homogeneous ooplasm (Fig. 1b,f), whereas Golgicomplexes and RER networks resided in the strands centers(Fig. 1b). As mid-previtellogenesis progressed, the granularooplasm expanded toward the oocyte plasma membrane.Concurrently, the strands of the granular ooplasm becameprogressively thinner and more convoluted. Finally, inyoung stage 3 oocytes, both types of ooplasm blendedtogether and were no longer recognizable as separateentities (Fig. 2a). At the same stage, the lipid bodydispersed, leading to the uniform distribution of lipiddroplets throughout the ooplasm (Fig. 2a).

Stage 3 oocytes (120–250 μm in diameter) had largegerminal vesicles containing, as in Xenopus, numerous denseperipherally located nucleoli and less dense Cajal bodies(Fig. 2a). At this stage of oogenesis, sections of lampbrushchromosomes were also recognizable (Fig. 2c, arrowheads)in the karyoplasm. The nuclear envelope formed longradially extending projections (Fig. 2c,d; asterisks); it wasperforated by numerous pore complexes and associated withmitochondria (Fig. 2d). The aggregates of nuage lost directcontact with the mitochondria and formed irregular “sponge”accumulations often intermingled with elements of RER(Figs. 1f, 2d,e). Such accumulations were observed in thevicinity of the nuclear envelope (Fig. 2d) and in moreperipheral ooplasm (Fig. 2e).

The morphological results described in this section clearlyindicated that the lipid body present in stage 1 and stage 2

oocytes did not represent a true Balbiani body. Continuing oursearch for the presence of a putative equivalent or homolog ofthe Balbiani body in sturgeon oocytes, we performedmolecular analyses of previtellogenic oocytes. We usedantibody against the RNA-binding protein Vera and antisenseRNA probes recognizing RNAs localized within the XenopusBalbiani body/germ plasm and Drosophila polar plasm(equivalent to Xenopus germ plasm) to determine whethertheir homologs were present in sturgeon Balbiani ooplasm.

Distribution of Balbiani body/germ plasm moleculesin sturgeon oocytes

Using antisense RNA probes for two related RNAs(Drosophila nanos and its Xenopus homolog, Xcat2), wefound that putative sturgeon homologs of these RNAswere located exclusively in the granular ooplasm. In stage1 and 2 oocytes, these RNAs were concentrated aroundthe germinal vesicle and in the strands of the cytoplasmradiating toward the oocyte plasma membrane (Fig. 3a,e).In agreement with the results described in the previoussection, these strands became gradually thinner duringmid-previtellogenesis (stage 2) and finally disappeared(Fig. 3b,c). As a result, the ooplasm of stage 3 oocyteswas almost uniformly labeled with these probes (Fig. 3d).We used antisense probes for other distinct molecularmarkers of Xenopus Balbiani bodies (and germinal gran-ules and nuage), including Xpat and Xdazl RNAs, andfound that sturgeon oocytes were negative for the presenceof Xdazl RNA (Fig. 3f) but positive for RNA homolo-gous to Xpat RNA (Fig. 3g). The distribution of this RNAtype mimicked the pattern described for Xcat2/nanoshomologs (cf. Fig. 3a,e,g). Thus, at least two homologsof Xenopus Balbiani body RNAs were present in thegranular ooplasm of sturgeon oocytes. However, becausethe RNA probes used in these study were deliberatelyhydrolyzed to short fragments, these probes might havedetected sturgeon RNAs that had only a short fragmenthomologous (and no direct homology) to Xenopus orDrosophila transcripts.

The presence, in sturgeon granular ooplasm, of RNAsputatively related to Xenopus Balbiani body RNAssuggests that the granular ooplasm is equivalent to theXenopus Balbiani body. However, in contrast to Xenopusand teleost Balbiani bodies, the sturgeon granular ooplasmis not restricted to a distinct cytoplasmic body butsurrounds the entire germinal vesicle. As described inthe previous section, the granular ooplasm also containsmitochondria, elements of RER, Golgi complexes, nuageaggregates, and nuage/mitochondria complexes, all ofwhich are characteristic of the Balbiani bodies of variousinvertebrates and vertebrates, including Xenopus andteleost fish.

R Fig. 1 Stage 1 and 2 oocytes: morphology and ultrastructure. a Stage1 oocyte with its surrounding thick basal lamina (bl), germinal vesicle(gv), homogeneous ooplasm (ho), granular ooplasm (dotted outline),accumulation of lipid droplets (lb lipid body), and nucleoli (nu). Semi-thin section stained with methylene blue. b Fragment of stage 2oocyte. Two distinct zones of ooplasm are clearly recognizable (erendoplasmic reticulum, Gc Golgi complex, m mitochondria, hoislands of homogeneous ooplasm). Semi-thin section stained withmethylene blue; differential interference contrast (DIC). c Stage 1oocyte. Fragment of a germinal vesicle with adjacent ooplasm (nunucleolus, arrowheads initial accumulations of nuage, arrows nuageaccumulation in contact with mitochondria and endoplasmic reticulumvesicles, m mitochondria, er endoplasmic reticulum). Electronmicrograph (EM). d Stage 1 oocyte. Fragment of a perinuclearooplasm with accumulations of nuage in contact with mitochondria(m) and endoplasmic reticulum elements (er). EM. e–g Stage 2 oocyte.EMs. e, g Granular ooplasm with endoplasmic reticulum (er), Golgicomplex (Gc), and nuage accumulations surrounded by mitochondriaand endoplasmic reticulum vesicles (arrow). f Contact zone (dottedline) between homogeneous (ho) and granular (go) ooplasm (ld lipiddroplets, m mitochondria, arrow nuage accumulations surrounded bymitochondria and endoplasmic reticulum vesicles, double arrowsponge-like nuage accumulation, er endoplasmic reticulum). Bars10 μm (a, b), 1 μm (c–g)

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In Xenopus oocytes, the Vg1-RNA-binding protein Vera,which is a highly conserved RNA-binding protein in variousvertebrate species (Deshler et al. 1997), has been reported topromote RNA localization via association with the endo-plasmic reticulum and/or cellular cytoskeleton (Deshler et al.1997, 1998). Because the granular ooplasm of the sturgeoncontained a prominent network of the endoplasmic reticulum(Figs. 1b,f, 2e), we investigated whether the homolog of theVera protein was present in this ooplasm. Using an antibodyagainst Xenopus Vera, we found that the sturgeon homologof this protein was highly concentrated in the strands of

granular cytoplasm throughout previtellogenesis (Fig. 3h).This suggested that the granular ooplasm of sturgeon oocytescorresponded to the Xenopus Balbiani body not onlystructurally, but also functionally, possibly participating inRNA transport or localization.

We also immunostained sturgeon oocytes with anti-Fatvg antibody against Xenopus Fatvg protein. Fatvgbelongs to a family of highly conserved proteins thatincludes mouse adipose differentiation related protein(ADFP) and human adipophilin (Chan et al. 1999). Fatvgis present in the fat body and also in the oocyte cytoplasm

Fig. 2 Stage 2 and 3 oocytes:morphology and ultrastructure.a Stage 3 oocyte. Perinuclearooplasm and germinal vesicle(gv) containing nucleoli andCajal bodies (small arrows);note the uniform distribution oforganelles and lipid droplets inthe ooplasm. Semi-thin sectionstained with methylene blue.b Stage 2 oocyte. Fragment of alipid body (ld lipid droplets).EM. c Stage 3 oocyte. Fragmentof a germinal vesicle and adja-cent ooplasm (Cb Cajal body, nunucleoli, arrowheads sectionedlampbrush chromosomes, aster-isks projections of nuclear en-velope). Semi-thin sectionstained with methylene blue. dStage 3 oocyte. Fragment of agerminal vesicle with adjacentooplasm (asterisk projection of anuclear envelope, m mitochon-dria, nu nucleolus, pc porecomplexes, double arrowsponge-like nuage accumula-tion). EM. e Stage 3 oocyte.Sponge-like nuage accumulationin contact with elements ofendoplasmic reticulum (er).Note also the mitochondria (m)and lipid droplets (ld). EM. Bars10 μm (a, c), 1 μm (b, d, e)

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on the surface of lipid vesicles (Chan et al. 2001; Chan etal., unpublished). We found that, in sturgeon oocytes, Fatvgprotein, in contrast to Vera protein, was distributedthroughout the cytoplasm in stage 1, 2, and 3 oocytes andwas also concentrated around lipid droplets (Fig. 3i).Control immunostaining performed without primary anti-bodies (anti-Vera or anti-Fatvg) were negative (not shown).

Discussion

We have found that the accumulation of lipid droplets (termedhere “the lipid body”) present in sturgeon oocytes does notcontain organelles or RNAs characteristic ofXenopus Balbiani

bodies and thus cannot be interpreted as an equivalent of thisorganelle. However, our studies indicate that the granularooplasm of sturgeon oocytes and Xenopus Balbiani bodiesshare important similarities (homologies) reflected in theirmolecular composition and ultrastructure. These include thepresence of nuage and of potential homologs of variousBalbiani body-associated RNAs and an abundance ofendoplasmic reticulum. Since granular ooplasm in sturgeonis not restricted to a specific oocyte region and does not forma distinct body (as does the Balbiani body), it will be termedhere the “Balbiani cytoplasm.”

In stage 1 sturgeon oocytes, the Balbiani cytoplasm isconcentrated around the germinal vesicle. As oogenesisprogresses, the Balbiani cytoplasm expands toward the

Fig. 3 In situ hybridization andimmunostaining. a–d Stage1–3 oocytes. Hybridizationwith antisense nanos RNAprobe; nanos-like RNA is pres-ent in strands of granularooplasm (stages 1 and 2) and inthe entire ooplasm (stage 3).e Stage 1 oocyte. Hybridizationwith antisense Xcat2 probe.f Stage 1 oocyte. Hybridizationwith antisense Xdazl probe.g Stage 1 oocyte. Hybridizationwith antisense Xpat probe.h Stage 1 oocyte. Immunolocal-ization of Vera protein. i Stage 3oocyte. Immunolocalizationof Fatvg protein; Fatvg proteinis concentrated around lipiddroplets. Bars 10 μm

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oocyte periphery and gradually blends in with the homo-geneous (organelle-free) ooplasm; this process is schemat-ically shown in Fig. 4. The expansion leads to the uniformdistribution of RNAs and nuage aggregations that wereinitially concentrated within the Balbiani cytoplasm(Fig. 4). Interestingly, the morphology and distribution ofthe Balbiani cytoplasm in sturgeon oocytes are similar tothat of basophilic cytoplasm surrounding the germinalvesicle in the oocytes of Oryzias latipes (Kobayashi andIwamatsu 2000); this indicates that the presence of Balbianicytoplasm is not limited to sturgeon oocytes but is probablya common feature of the oocytes of other fish species.

We suggest that, in sturgeon, the expanding Balbianicytoplasm is responsible for the transportation of variousRNAs (including possible homologs of Xcat2/nanos andXpat) and germ cell determinants (nuage) from the vicinityof the germinal vesicle toward the oocyte periphery.Interestingly, the delivery of germ cell determinants(germinal granules) and localized RNAs to the vegetalcortex in Xenopus oocytes has previously been suggestedalso to rely on the expansion of the Balbiani body (Wilk etal. 2005). Although the mechanism responsible for theexpansion of the Balbiani body in Xenopus and of theBalbiani cytoplasm in sturgeon oocytes remains uncharac-terized, we are tempted to speculate that, during vertebratephylogeny, similar cellular machinery has been adopted totransport RNAs and germ cell determinants from thevicinity of the germinal vesicle either to disperse them inthe oocyte cytoplasm (sturgeons) or to localize them to aspecific region of the oocyte (vegetal cortex in Xenopus).

According to a recent hypothesis, some of the Balbianibody RNAs (including Xcat2 and Xpat) of Xenopus arelocalized to the Balbiani body through a diffusion/entrap-ment mechanism and association with the endoplasmic

reticulum and the RNA-binding protein Vera, whichcolocalizes with the endoplasmic reticulum (Chang et al.2004; Deshler et al. 1997, 1998). Since, in sturgeonoocytes, the putative homologs of Balbiani body RNAsand the RNA-binding protein Vera are present in the regionoccupied by an elaborate network of endoplasmic reticulumwithin the Balbiani cytoplasm, we suggest that, in stur-geons, the homologs of Balbiani body RNAs are alsoassociated and possibly transported via interaction withVera and the endoplasmic reticulum.

Studies of Xenopus oocytes have clearly indicated that atleast some Balbiani body RNAs are localized inside and/orassociated with the germinal granules or nuage (Kloc et al.2002). These RNAs include Xcat2 (present in nuage andgerminal granules) and Xpat and DeadSouth (both associatedwith the periphery of germinal granules). Because we havestudied the distribution of putative homologs of these RNAsin the Balbiani cytoplasm of sturgeon oocytes only at thelevel of the light microscope, we do not know whether theseRNAs colocalize with the nuage. Although the abundance ofvarious forms of nuage in the Balbiani cytoplasm of sturgeonoocytes suggests that homologs of Xenopus Balbiani bodyRNAs are associated with sturgeon nuage, a definitiveanswer has to await the results of the identification andcloning of sturgeon homologs of Xenopus transcripts and insitu hybridization studies at the electron-microscopy level.

Acknowledgements The authors are grateful to Prof. R. Kolman(Institute of Inland Fisheries, Olsztyn-Kortowo, Poland) who sharedhis passion and knowledge of sturgeons with us and who performedthe ovarian biopsies, to the staff of the Institute of Ichthyology andFisheries (Agricultural Academy, Krakow, Poland) for the specimensof Acipenser gueldenstaedtii, to Prof. E. Pyza (Institute of Zoology,Jagiellonian University, Krakow, Poland) for electron microscopyfacilities, and to Mrs. B. Szymanska, Mrs. W. Jankowska, and Mrs. E.Kisiel for technical assistance.

Fig. 4 Distribution of homoge-neous (yellow) and granular(violet) cytoplasm in threeconsecutive stages of oocyte de-velopment. a Stage 1. b Stage 2.c Stage 3; in this stage both typesof the ooplasm blend together andare no longer recognizable asseparate entities. Nuage accumu-lations (black dots) are initially(a) distributed around the germi-nal vesicle (white). As oogenesisprogresses, they are transportedtoward the oocyte periphery(b) and finally become uniformlydispersed throughout the entireooplasm (c)

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