Developmental pattern of telomerase expression in the sand scallop, Euvola ziczac

Developmental pattern of telomerase expression in the sand scallop, Euvola ziczac

Richard Owen,1,2 Samia Sarkis,3 and Andrea Bodnar1,a

1 Bermuda Biological Station for Research, Ferry Reach, St. George’s GE 01, Bermuda2 School of Biological Sciences, University of Plymouth, Portland Square, Plymouth, PL4 8AA, UK

3 Department of Conservation Services, Flatts, Bermuda

Abstract. Telomerase is a ribonucleoprotein that can maintain telomeres, the repetitivesequences of DNA found at the end of eukaryotic chromosomes, and confer long-termproliferative capacity on cells. Telomerase expression is essential during periods of intense celldivision such as the early developmental process. In later development, some species retaintelomerase activity while others repress telomerase activity in what is thought to be a tumor-protective mechanism. Despite the importance of telomerase expression in both developmentand neoplastic disease, no studies to date have characterized its expression in bivalves. Wepresent the first report of telomerase expression in a bivalve species, the sand scallop, Euvolaziczac. Telomerase activity was detected throughout the early stages of development and in alladult tissues examined. Analysis of DNA isolated from adult tissues indicated long telomeres,with terminal restriction fragment lengths 420 kb in both somatic and germ tissues. Ubi-quitous telomerase expression throughout development and into adulthood would suggest alack of telomere-related senescence and suggests that these scallops do not use telomeraserepression as a mechanism to suppress the formation of neoplasm.

Additional key words: telomere, development, bivalve, mollusc

Telomeres are the repetitive sequences of DNAand associated proteins that cap the ends of eukary-otic chromosomes and play an essential role in main-taining genome stability (Campisi et al. 2001; Feldseret al. 2003). Telomeres shorten during cell divisiondue to the inability of the DNA replication machin-ery to replicate the extreme end of the linear DNAmolecule (Campisi et al. 2001). When telomeres be-come critically short, they trigger a signal for the cellto permanently stop dividing, a process referred to ascellular senescence (Campisi et al. 2001). The onsetof senescence prevents the erosion of essential geneslocated near telomeres and prevents catastrophic ge-nomic instability (Campisi et al. 2001; Feldser et al.2003). Immortal cells and cells that require a highreplicative potential have evolved a number of strat-egies to counteract this progressive loss of telomeres,including recombination and retrotransposition,but the most common solution is expression of anenzyme called telomerase. Telomerase is a ribonu-cleoprotein that can synthesize telomeric DNA and

replace that which is lost with each round of DNAreplication (Campisi et al. 2001). Telomerase expres-sion is widely conserved in eukaryotic evolution.Although there have been few studies in marineorganisms, it has been detected in numerous species,including protozoa, yeast, plants, and mammals,where it has been shown to be necessary to maintainstable telomeres and allow sustained cell division(Greider 1990; Campisi et al. 2001; Forsyth et al.2002).

Telomerase expression is particularly important tooffset telomere loss during periods of extensive cellproliferation such as during the developmental proc-ess. In human development, telomerase is present inearly embryogenesis and in all tissues examined dur-ing the first trimester; however, it is repressed at dif-ferent times as fetal development progresses (Forsythet al. 2002). In the absence of telomerase, telomeresshorten in somatic tissues with consecutive cell div-isions during growth into adulthood until the onsetof cellular senescence (Campisi et al. 2001; Forsythet al. 2002). Telomerase remains active in germ cellsand cell types that require a high proliferative capac-ity (i.e., stem cells) and is reactivated during the pro-gression of most cancers. In addition to humans,

Invertebrate Biology 126(1): 40–45.

r 2007, The Authors

Journal compilation r 2007, The American Microscopical Society, Inc.

DOI: 10.1111/j.1744-7410.2007.00074.x

aAuthor for correspondence.

E-mail: [email protected]

telomerase inactivation has been observed in otherlarge mammals and some avian species (Forsyth et al.2002; Argyle et al. 2003). It is thought that telomeraseinactivation during development has evolved as a‘‘tumor-protection mechanism’’ that reduces the in-cidence of cancer in large, long-lived organisms(Campisi et al. 2001; Forsyth et al. 2002). In con-trast, other organisms such as mouse, most plants,and some marine vertebrates and invertebrates(sponges, lobsters, fish, sea cucumbers, and eels) donot repress telomerase activity in later development(Fitzgerald et al. 1996; Klapper et al. 1998; Forsythet al. 2002; McChesney et al. 2004).

Scallops are important members of marine ecosys-tems and are commercially important species. Theyhave been harvested since colonial times, and reportsof the scallop fishing industry and the life history ofsome species of scallops date back to 1910 (Belding1910). Since then, the pattern of development,growth rate, and survival has been the subject ofmany studies (Sastry 1965; Costello et al. 1973;Bourne et al. 1989; Couturier et al. 1996; Sarkiset al. 2003). An understanding of the factors influen-cing both development and neoplastic disease isimportant, especially in commercially important spe-cies. Despite the importance of telomerase expressionin both development and neoplastic disease, no stud-ies to date have characterized its expression inbivalves. In this study, we investigated the develop-mental pattern of telomerase expression in the sandscallop, Euvola ziczac LINNAEUS 1758.

Methods

Larval rearing and early developmental stages

Mature individuals of Euvola ziczac were collectedfrom the field and transported to the hatchery at theBermuda Biological Station for Research. Massspawning was induced immediately upon arrival byexposure to a series of thermal shocks as described inSarkis et al. (2003). Females were cross-fertilized withtwo or three males (1mL of sperm/1L of eggs). Fer-tilized eggs were passed through a 150-mm sieve anddistributed into 1000-L tanks at a density of10 eggsmL�1. Eggs were reared in 1-mm-filtered sea-water at a water temperature higher than ambient(T5 241C) and ambient salinity (S5 36ppt). Slightaeration was provided into the larval tanks. Feedingwas initiated 24h after fertilization and was com-posed of a mixture of live algal species. The rationwas increased throughout the duration of larval lifeas described in Sarkis et al. (2003). Water was chan-ged three times a week. Larvae were collected on

sieves; increasing mesh size was used as the larvaegrew, ranging from 60mm for D-larvae to 120mm forlate veliger larvae. Collected larvae were gentlywashed into holding containers during the time oftransfer. Larval tanks were cleaned, rinsed, and re-filled with heated seawater. Moribund larvae werediscarded, and larval cultures were passed through a300-mm sieve for removal of debris, before resuspen-sion into the larval tanks. The density of larvae de-creased as larvae grew, ranging from 8 larvaemL�1 atthe start of the larval cycle to 4 larvaemL�1 for lateveligers.

For the purpose of this work, larvae were rearedfor 9 d after fertilization to the late veliger stage,showing a developing umbone before the develop-ment of a foot. Samples at various developmentalstages, including second polar body formation(B40min post-fertilization), a multi-cellular stage(B6 h post-fertilization), D-larvae (2 d post-fertiliza-tion), and late veliger larvae (9 d post-fertilization),were taken from the holding containers, pelletedby centrifugation, washed briefly with TE buffer(10mM Tris, 1mM EDTA, pH 7.5), and frozen at�701C before analysis.

Collection of adults and preparation of tissues

Three sand scallop adults were collected from aseagrass bed in Harrington Sound, Bermuda. Shellheight, defined as the greatest distance between theanterior and the posterior end, was 68, 75.9, and71.8mm for each individual, respectively. The adultscallop tissues (adductor muscle, gill, mantle, andboth male and female gonads) were collected bydissection, briefly rinsed with TE buffer (10mMTris, 1mM EDTA, pH 7.5), and frozen at �701Cbefore analysis.

Telomeric Repeat Amplification Protocol Assay

Telomerase activity was determined with theTelomeric Repeat Amplification Protocol (TRAP)assay (Kim et al. 1994) using the TRAPeze kit fromChemicon International (Temecula, CA, USA). Inthis assay, telomerase activity is detected by its abilityto synthesize telomeric repeats onto an oligonucleo-tide substrate. These elongation products are thenamplified by PCR and separated by non-denaturingpolyacrylamide gel electrophoresis. The presence oftelomerase activity is evident by the appearance of aladder pattern of bands incremented by the telomericrepeat unit.

Detergent extracts were prepared from the scal-lop cells and tissues using 3-[(3-cholamidopropyl)

Telomerase expression in scallops 41

Invertebrate Biologyvol. 126, no. 1, winter 2007

dimethyl-ammonio]-1-propanesulfonate (CHAPS)lysis buffer containing 100UmL�1 of RNasin (Pro-mega, Madison, WI, USA), following the protocolsupplied with the TRAPeze kit. The eggs, sperm, andearly developmental stages were resuspended inCHAPS lysis buffer for extraction while the adult tis-sues were homogenized in CHAPS lysis buffer. Proteinestimations were performed using Coomassie proteinassay reagent from BioRad (Hercules, CA, USA), andthe CHAPS extracts were aliquotted and stored at�701C. For all TRAP reactions, the telomerase exten-sion step before PCR was increased to 60min andPCR amplification was performed for 33 cycles. ThePCR products were run on 12.5% non-denaturingpolyacrylamide gels and visualized using the DNAstain SYBR green (Molecular Probes, Eugene, OR,USA). The molecular weight of each band on the gelswas calculated using the Kodak Molecular ImagingSoftware (version 4; Kodak, New Haven, CT, USA).In this analysis, the molecular weights of the telomer-ase-generated bands were normalized to the molecularweight of the internal control bands to compensate forany curvature of the gels.

Isolation of DNA and telomere length analysis

DNA was extracted from scallop tissues using themethod of de Jong et al. (1998). Telomere length wasdetermined using terminal restriction fragment(TRF) length analysis, with the TeloTAGGG kitfrom Roche Applied Sciences (Indianapolis, IN,USA) and 1mg of DNA per lane. Genomic DNAwas digested with restriction enzymes that digest thegenome at frequent sites, but do not digest the DNAwithin the telomeric repeat sequence. The digestionproducts were run on a 0.8% agarose gel, transferredto a nylon membrane, and probed with an oligonu-cleotide probe complementary to the telomeric se-quence TTAGGG.

Results

Telomerase activity: early developmental stages

Mature eggs were found to have telomerase activ-ity while telomerase could not be detected in maturesperm (Fig. 1). Mature sperm were assayed using anextended range of protein concentrations (0.03–1.2mg protein added to the TRAP assay), but no tel-omerase activity was detected (unpubl. data). EachTRAP reaction mixture contains primers and a tem-plate for amplification of a 36-bp internal control,which helps to identify false-negative samples thatcontain inhibitors of Taq polymerase. Inhibitors ofTaq polymerase would prevent the amplification of

telomerase-generated products in the TRAP assay.The presence of the internal control band in thesperm samples indicates the absence of a Taq polym-erase inhibitor in these samples (Fig. 1). Followingfertilization, samples of early developmental stages,including the formation of the second polar body(B40min post-fertilization), a multi-cellular stage(B6 h post-fertilization), D-larvae (2 d post-fertiliza-tion), and late veliger larvae (9 d post-fertilization),were collected. All stages of early development inves-tigated showed telomerase activity, as seen by theladder pattern of bands on the gel following theTRAP assay (Fig. 1). The molecular weights of thebands generated by the TRAP assay were calculatedusing the Kodak Molecular Imaging Software (ver-sion 4). This analysis indicated that the periodicity ofthe bands for scallops was identical to that generatedby the human transformed cell line 293, which wasused as a positive control and which possesses thetelomeric repeat sequence TTAGGG. The specificityof the telomerase reaction was confirmed by the lackof the ladder pattern of bands in the extracts thatwere heat treated (851C for 10min) before perform-ing TRAP analysis.

Telomerase activity: adult tissues

CHAPS detergent extracts were prepared fromvarious tissues of three adult scallops and assayedfor telomerase activity using the TRAP assay. Tel-omerase activity was detected in the tissue samplesexamined including adductor muscle, gill, mantle,and both male and female gonads (Fig. 2). Themolecular weights of the bands calculated by theKodak Molecular Imaging Software (version 4)were identical to those generated by the positive con-trol 293 cells. Further, when dCTP was omitted fromthe telomerase extension portion of the TRAP reac-tion, it did not affect the result, indicating the absenceof dCTP in the scallop telomeric sequence (unpubl.data).

Telomere length analyses

The majority of telomeric signals from scallopDNA was beyond the limit of resolution of thisTRF analysis (420kb) for all somatic and germtissues (Fig. 3).

Discussion

We present the first report of expression of telom-erase activity throughout early development and inadult tissues of a bivalve species, the sand scallop,Euvola ziczac. Telomerase activity and telomere

42 Owen, Sarkis, & Bodnar


length were detected using commercially availablekits that have been optimized to the human telomer-ic sequence TTAGGG. This telomeric sequence ishighly conserved in both vertebrates and inverte-brates, including bivalve molluscs such as the wedge-shell clam (Donax trunculus LINNAEUS 1758), themussel (Mytilus galloprovincialis LAMARCK 1819),and the bay scallop (Argopecten irradians DALL

1898) (Estabrooks 1999; Plohl et al. 2002). The peri-odicity of the telomerase-generated bands of E. zic-zac indicated a six-nucleotide repeat sequence, andomission of dCTP from the telomerase extensionportion of the TRAP reaction indicated the absenceof dCTP from this scallop’s telomeric sequence.These results suggest that the telomeric sequence ofE. ziczac is similar to that of other bivalves.

Telomerase activity was found in mature eggs butnot in mature sperm of the scallop. The presence oftelomerase activity in mature eggs is in contrast toobservations in other species (i.e., humans) wheremature eggs do not express telomerase (Forsyth et al.2002). This suggests that there would be nodelay between the start of the developmental proc-ess and the expression of telomerase in scallops. Tel-omerase was detected in both the male and femaleparts of the gonad, where it is presumably necessaryto maintain telomere length during gametogenesis ashas been described for other species (Forsyth et al.2002). The presence of telomerase activity in all adulttissues examined indicates that scallops do not re-press telomerase in later development. Telomerase

repression during development has been observed insome avian species, humans, and other large mam-mals, whereas other mammals (i.e., mouse) and mostplants do not repress telomerase and appear to main-tain stable telomeres during development and growth(Fitzgerald et al. 1996; Forsyth et al. 2002; Argyleet al. 2003). In aquatic species, telomerase activity hasbeen detected in differentiated tissues of sponges, lob-sters, sea cucumbers, eels, and fish (Klapper et al.1998; Koziol et al. 1998; McChesney et al. 2004). Ithas been suggested that aquatic animals that growthroughout their lifespan require telomerase activityto maintain telomeres during this continual growth(Klapper et al. 1998).

Telomere lengths were not measured in the devel-opmental stages of E. ziczac but in adult tissues, todetermine whether telomeres were maintained duringgrowth of this scallop. Telomere lengths, measured by

Fig. 1. Early developmental stages in

Euvola ziczac and associated

telomerase activity. A. Embryonic

and larval developmental stages

including fertilized egg (egg), polar

body (pb), multi-cellular stage

(multi), D-larva (dl), and late

veliger larva (lvl). B. Telomerase

activity in early developmental

stages analyzed by the telomeric

repeat amplification protocol

(TRAP) assay. Lanes 1–3, sperm;

lanes 4–6, unfertilized eggs; lanes 7–

9, second polar body; lanes

10–12, multi-cellular stage; lanes

13–15, D-larvae; lanes 16–18, late

veliger larvae; lane 19, lysis buffer

(lb) control; lanes 20–21, positive

control 293 cells (1000-cell equiva-

lents). In lanes 1, 4, 7, 10, 13, and

16, the amount of 3-[(3-chol-

amidopropyl) dimethyl-ammonio]-

1-propanesulfonate (CHAPS) ex-

tract added to the TRAP reaction

was 0.4mg protein. In lanes 2, 5, 8,

11, 14, and 17, the amount of

CHAPS extract added to the TRAP

reaction was 0.1mg protein. In lanes

3, 6, 9, 12, 15, and 18, the CHAPS

extracts (0.4mg protein) were heat

inactivated before TRAP analysis

(hi). In lane 21, the 293 CHAPS

extract (1000-cell equivalents) was

heat inactivated before TRAP

analysis (hi). The internal control

for the TRAP reaction is indicated

with the arrow (ic).



TRF analyses, were long (420kb) in both somaticand germ tissues, suggesting maintenance of telom-eres. However, as the telomere length values werebeyond the limit of resolution of the TRF analyses,small changes in telomere length between somaticand germ tissues would not be distinguished usingthis analysis. Alternative methods for measuring tel-omere length must be used to confirm this result.

Quantitation of the relative levels of telomerase indifferent developmental stages and adult tissues wasnot performed in this initial study. Quantitation iscomplicated by the possibility that individual cellswithin a tissue could express different levels of tel-omerase or that telomerase expression may be con-fined to actively dividing cells. There is some evidenceof telomerase regulation in sponges, where telomer-ase is present in dividing tissues but not in dissoci-ated, non-dividing sponge cells (Koziol et al. 1998).Telomerase regulation has also been observed in non-autonomous catfish leukocyte cell lines (Barker et al.2002). In addition, most plants display regulated tel-omerase activity where it is present in undifferentiat-ed cells and absent from non-proliferating tissues(Fitzgerald et al. 1996). Quantitation of telomerase

activity in scallop tissues may be of value in futurestudies, to assess whether activity is altered withenvironmental conditions that affect overall growthor in comparing healthy and neoplastic portionsof a tissue.

Maintenance of telomeres by the activation oftelomerase or other mechanisms is necessary to con-fer immortality on cells, and telomerase is expressedin the vast majority of tumors in humans andother species (Kim et al. 1994; Bodnar et al. 1998).In humans and other large mammals, telomerase re-pression is thought to be a ‘‘tumor-protection mech-anism’’ that would suppress the formation ofneoplasm in these large, long-lived species. Thisimplies that cells that do not repress telomeraseare more susceptible to immortalization, as has beenobserved in cultured mouse cells (Forsyth et al. 2002).Although neoplasm is common in some species of bi-valves, there are few reported cases in scallops (Barber2004). In the Registry of Tumors in Lower Animals(RTLA) (www.pathology-registry.org), there are 221cases reported for softshell clams, 58 cases for hardcalms, 79 cases for mussels, and only one case for a

Fig. 2. Analysis of telomerase activity in adult tissues of

Euvola ziczac. Lanes 1–2, adductor muscle (ad) (2mgprotein); lanes 3–4, gill (2mg protein); lanes 5–6, mantle

(man) (2mg protein); lanes 7–8, female gonad (fg) (0.4mgprotein); lanes 9–10, male gonad (mg) (0.4mg protein);

lanes 11–12, positive control 293 cells (1000-cell

equivalents). In lanes 2, 4, 6, 8, 10, and 12, the 3-[(3-

cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate

(CHAPS) extracts were heat inactivated before telomeric

repeat amplification protocol (TRAP) analysis (hi). The

internal control for the TRAP reaction is indicated with the

arrow (ic).

Fig. 3. Terminal restriction fragment length analysis of

DNA isolated from adult tissues of Euvola ziczac. Lane 1,

molecular weight markers (mr); lane 2, adductor muscle

(a); lane 3, gill (g); lane 4, mantle (m); lane 5, female gonad

(f); lane 6, male gonad (mg).

44 Owen, Sarkis, & Bodnar


scallop species (Pecten sp.). It would be interesting tocompare not only telomere biology but also cellulardefense and repair mechanisms across these bivalvespecies to better understand this apparent differingsusceptibility to acquiring neoplasm.

Acknowledgments. We would like to thank LucyBuxton, Nicole Allard, and Nicola Francis for theirassistance with the experiments. We are grateful for thefinancial support from the Ray Moore Endowment Fundand the Duperreault Ocean Genomics Fund at BBSR,which made this work possible. This manuscript has beenassigned BBSR contribution number 1679.

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Developmental pattern of telomerase expression in the sand scallop, Euvola ziczac