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Insect Biochemistry and Molecular Biology 41 (2011) 823e831

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Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier .com/locate/ ibmb

Transcriptome and gene expression profile of ovarian follicle tissueof the triatomine bug Rhodnius prolixus

Marcelo N. Medeiros a,g, Raquel Logullo b,g, Isabela B. Ramos a,g, Marcos H.F. Sorgine c,g,Gabriela O. Paiva-Silva c,g, Rafael D. Mesquita d,g, Ednildo Alcantara Machado a,g,Maria Alice Coutinho b,g, Hatisaburo Masuda c,g, Margareth L. Capurro e,g, José M.C. Ribeiro f,Glória Regina Cardoso Braz b,g,*,1, Pedro L. Oliveira d,g,1

a Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, BrazilbDepartamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Brazilc Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazild Instituto Federal de Educação do Rio de Janeiro, Rio de Janeiro, RJ, BrazileDepartamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazilf Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases,12735 Twinbrook Parkway, Room 2E32, Rockville, MD 20852, USAg Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil

a r t i c l e i n f o

Article history:Received 17 March 2011Received in revised form13 June 2011Accepted 16 June 2011

Keywords:Reticulum stressRhodnius prolixusTranscriptomeChorionOocyteApoptosis

* Corresponding author. Av. Athos da Silveira RamoCidade Universitária e Ilha do Fundão, Rio de JaneirTel.: þ55 21 2562 7365; fax: þ55 21 2562 7266.

E-mail address: gbraz@iq.ufrj.br (G. R. Cardoso Bra1 These authors contributed equally to this work.

0965-1748/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.ibmb.2011.06.004

a b s t r a c t

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts theoocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describea transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase ourknowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library ofdifferent stages of follicle development. The transcriptome showed high commitment to transcription,protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich proteinwith maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We alsofound Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that wasan eggshell component candidate. Six transcripts coding for proteins related to the unfolded-proteinresponse (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPRshowed higher expression during early stages of development and downregulation during late stages,when transcripts coding for S proteins participating in chorion formation were highly expressed. Severaltranscripts with potential roles in oogenesis and embryo development are also discussed. We proposethat intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression ofa set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation.This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting modelfor studying control of RS and cell survival in professional S cell types.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Making viable eggs that will survive in the environment inde-pendently of the maternal body is a key step in the reproductionprocess of all oviparous animals, including insects. In their way

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from oocyte precursor to the mature eggda process frequentlycompleted in just a few hours, these cells accumulate largeamounts of reserves and increase about a thousand-fold in size,followed by isolation from the external world by formation of theeggshell. Egg development in insect ovaries is divided into threemain phases: pre-vitellogenic growth, vitellogenic growth (alsocalled vitellogenesis), and choriogenesis. Pre-vitellogenic growthoccurs in the anterior end of the ovariole, in the case of thehemipteran telotrophic ovary, in a lanceolate structure calledtropharium (Atella et al., 2005). Despite their astonishing growthrates, oocytes themselves show very modest synthetic activity(Wallace et al., 1972), and their development is supported mainly

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by other cells either from the ovary or from extra-ovarian tissues(i.e., fat body).

Early growth of oocytes takes place in close association withother cell types such as the surrounding follicular epithelium (FE)and nurse cells. These are linked to oocytes by means of cyto-plasmic projections/bridges called trophic cords that are used toprovide both proteins and RNA. Trophic cords are retained until theearly phases of vitellogenic growth (Telfer et al., 1982). Duringvitellogenesis, oocytes rapidly accumulate large amounts of lipids,glycogen, RNA, and yolk proteins. Vitellin, the most common yolkprotein, derives from a hemolymphatic lipoprotein called vitello-genin (Vtg), which in most cases is synthesized in the fat body andis taken up by oocytes by receptor-mediated endocytosis. Synthesisof Vtg by ovarian FE (OFE) cells has been reported in cycloraphandipteran ovaries (Postlethwait et al., 1980) as well as in the tri-atomine Rhodnius prolixus (Melo et al., 2000). Also, non-vitellin yolkproteins such as the 30-kDa protein from lepidopteran eggs aresynthesized by FE. Upon completion of vitellogenesis, fully growneggs are separated from the external world by the chorion,a protective proteinaceous layer synthesized and secreted byfollicle cells at the very end of oocyte development inside the ovary.After choriogenesis, FE cells degenerate and die, exhibiting severalfeatures characteristic of programmed cell death, in a process thatis believed to be important to prevent blockage of the ovarioles andto support proper egg development (Nezis et al., 2006, 2002).

The endoplasmic reticulum (ER) is the gate to the exocytoticpathway, where newly synthesized secretory (S) proteins must foldand acquire their correct functional tri-dimensional structure. Inrecent years, it has been recognized that frequently a large portionof nascent polypeptide chains fail to fold appropriately, eventuallyleading to accumulation of misfolded proteins in the ERdwhat iscalled reticulum stress (RS) (Banhegyi et al., 2007). Eukaryotic cellsrespond to ER stress by increasing transcription of genes for ERresident chaperones and proteins that help either to accelerate fluxin the secretory pathway or to promote destruction of misfoldedproteins (Kohno et al., 1993; Mori et al., 1992; Romisch, 2005).Collectively, this process has been termed the unfolded-proteinresponse (UPR). To eliminate organelles or unhealthy cells,autophagy or apoptotic cell death take place whenever the UPRdoes not occur properly or is not sufficient to deal with the ER stresschallenge (Lai et al., 2007).

To provide insights into the molecular framework involved indevelopment of the insect ovary, we describe here a transcriptomeof the most abundant transcripts found in the follicle of the tri-atomine R. prolixus, a vector of Trypanosoma cruzi, the causativeagent of Chagas disease. Several messages related to oocyte andembryo development were identified, and expression of selectedtranscripts during follicle development was studied. We proposethat failure in triggering UPR during choriogenesis leads to death ofFE cells as part of the developmental program of the insect egg.

2. Materials and methods

2.1. Insects and tissues

R. prolixus were from a colony maintained at 28 �C and 70%relative humidity. Insects used here were adult mated femaleshaving their second blood meal after their imaginal molt. Ovarioleswere dissected free of tracheae and ovarian sheath. Ovarian folliclesat vitellogenic stages (0.5e2.0 mm) or initiating choriogenesis(about 2 mm) were opened with tweezers and the oocyte contentdiscarded. Due to their reduced size, pre-vitellogenic follicles wereseparated from tropharium and used both for library and quanti-tative real-time PCR (qPCR). Tropharium samples were used onlyfor qPCR and were not included in the cDNA library.

2.2. cDNA library and sequencing

mRNA was isolated from using the Micro FastTrack mRNAisolation kit (Invitrogen, Carlsbad, CA), and the PCR-based cDNAlibrary was made following the instructions for the SMART cDNAlibrary construction kit (Clontech, Palo Alto, CA) according to themanufacturer’s instructions. cDNA from follicle cells isolated asdescribed abovewere fractionated in three size intervals using a gelfiltration column, ligated separately to the l TriplEx 2 vector,packaged with packing extract Gigapack III Gold (Stratagene, LaJolla, CA), and then joined in a single pool. The obtained phagelibrary was mixed with log phase XL-1 Blue cells (Stratagene) andplated in agar medium containing IPTG and X-gal for blue/whitescreening. A sample of individual white lysis plates was chosenrandomly and the cloned insert amplified using PT2F1 (50-AAG TACTCT AGC AAT TGT GAG C-30, upstream) and PT2R1 (50 CTC TTC GCTATT ACG CCA GCT G-30, downstream) primers and sequenced ina DNA sequencing instrument ABI 377 (Applied Biosystems, FosterCity, CA) using the BigDye Terminator 3.0 kit (Applied Biosystems)and the primer PT2F3 (50-TCT CGG GAA GCG CGC CAT TGT-30,downstream a PT2F1) as described elsewhere (Valenzuela et al.,2002).

2.3. Bioinformatics

The sequences obtained were cleaned from vector and primersequences and assembled into contigs using CAP3 software (HuangandMadan,1999) mastered by an in-house bioinformatics program(Ribeiro et al., 2007). The protein coding potential of the contigsand singletons obtained was examined using the blastx algorithm(Altschul and Gish, 1996) to search the non-redundant protein (NR)and Gene Ontology (GO) (Ashburner et al., 2000) databases. As thetranslation mRNA of proteins that are coded by mitochondrial DNAinvolves the use of an alternate genetic code, we have alsocompared the transcripts with a database created from mitochon-drial DNA sequences. Essentially, these analyses were performedusing a program pipeline already described in the literature(Valenzuela et al., 2003). To get hints on the possible biologic role ofthe proteins obtained from the translated contigs, rpsblast wasused to search for conserved protein domains in the Pfam (Batemanet al., 2000), SMART (Schultz et al., 2000), COG (Tatusov et al.,2003), and conserved domains (CDD) (Marchler-Bauer et al.,2002) databases. This information is particularly important,because we kept in our analysis open-reading frames that lack theinitial methionine (50-truncated), stop codon (30-truncated), orboth (fragment) and not only full-length coding sequences.Although the BLAST result against GO gives insight to a putativefunction of the transcripts, in the process of manual annotation thetranscripts were classified into one of the following fifteen classes:protein synthesis, protein export machinery, transcriptionmachinery, transcription factor, proteasome machinery, extracel-lular matrix (ECM)/S protein/ovarian function, protein modificationmachinery, energy metabolism, signal transduction, nuclear regu-lation, transporter, cytoskeletal/cell adhesion, intermediatemetabolism, unknown (U), and unknown conserved.

To confirm that the expressed sequence tags (ESTs) derived fromthe Rhodnius genome, these were also BLASTed against theR. prolixus genome. Supercontigs from the R. prolixus assembledgenome (release 1.0.1, from January 2009) were downloaded fromhttp://genome.wustl.edu/genomes/view/rhodnius_prolixus/. TheFASTA files of these supercontigs were broken into 50-kb fragmentswith 10 kb from previous sequence, and a database was created.This procedure was taken because BLAST does not work well ifsubjects in the database are very long sequences (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD¼Web&PAGE_TYPE¼BlastDocs).

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This formatted database was used to blastn the ESTs to thegenome of the insect. The few ESTs not found in any genome tracefile were disregarded unless they code for a sequence very similarto a known protein.

After assembly and comparison to databases, sequences codingfor proteins larger than 50 amino acids (aa) and/or that showedbest matches to NR with e-values lower than 10�5 were furtheranalyzed. As polypeptides produced by the FE cells that arecomponents of the chorion must be secreted, we tried to identifytranslation products that were potentially secreted. Segments ofthe six-frame translations of the EST having a methionine in thefirst 100 predicted amino acids were submitted to the SignalPserver (Nielsen et al., 1997). Multiple alignments of proteins ofinterest were performed using ClustalW (Thompson et al., 2002).

2.4. qPCR

Total RNA was isolated using TRIzol reagent (Invitrogen) fromisolated tropharium or ovarian follicles that were either pre-vitellogenic, vitellogenic, or that had already entered chorio-genesis. RNA extracted from oocyte content of vitellogenic or cho-rionated oocytes was also extracted and analyzed together withprevious preparations. Tissues were obtained as described above.Total RNA was quantified in a spectrophotometer, and 1 mg wastreated with 1 Unit of DNase (RNAse free; Invitrogen) for 30 min at37 �C. Reactions were stopped by adding 1 ml of 20 mM EDTA andheating for 10 min at 65 �C. cDNA synthesis was performed fromthe Dnase-treated RNA using the high-capacity cDNA reversetranscription kit from Applied Biosystems.

All primers (Supplemental Table S3) were made using Primer3 (http://frodo.wi.mit.edu/primer3/) and Oligoanalyzer software(http://www.idtdna.com/analyzer/applications/oligoanalyzer/).

Before qPCR reactions, all primers pairs were tested by conven-tional PCR using a 40 cycle reaction (94 �C, 30 s denaturation; 60 �C,30 s annealing; 72 �C, 30 s e Taq extension) in an Applied Bio-systems 2400 thermocycler and analyzed in 2% agarose gel.

Real-time qPCR was performed in an AB 7500 (Applied Bio-systems) using power SYBR-green PCRmastermix. The comparativeCt method (Livak and Schmittgen, 2001; Pfaffl, 2001) was used tocompare changes in gene expression levels. Actin gene expressionwas used as endogenous control, using primers based on an actincDNA sequence described elsewhere (Ribeiro et al., 2004): RpAct F50-AGTAGCTGCATGGGT TGTAG-3 (forward) and RpAct R 50-CAACATACATTGCTGGACTG-30 (reverse) (Alves-Bezerra et al., 2010).

Fig. 1. Functional classification of transcripts from ovary follicular epithelium libraryThe 252 transcripts that matched a known protein database with an e-value < 10�5

were grouped in 13 major functional classes: Protein synthesis, Protein exportmachinery, Transcription machinery, Transcription factor, Proteasome machinery,Extracellular matrix/Secreted protein/ovarian function, Protein modificationmachinery, Energy Metabolism, Signal transduction, Nuclear regulation, Transporters,Cytoskeletal/cell adhesion, Intermediate Metabolism.

2.5. Apoptosis TUNEL assay

Vitellogenic follicles never showed evidence of cell death,a feature characteristic of the end of choriogenesis. Late follicleswere first screened by Trypan blue exclusion assay to check for thepresence of dead cells. The FEs of selected oocytes were carefullydissected, fixed for 1 h in 4% paraformaldehyde, and treated for theterminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) assay using the ApopTagPlus kit (Chemicon,Temecula CA) following manufacturer’s instructions. Briefly, thecells were treated with proteinase K (20 mg/ml) to pretreat thetissue, followed by incubations in 3% H2O2 to quench endogenousperoxidase. Samples were then incubated for 2 h at 37 �C ina solution of terminal deoxynucleotidyl transferase. After washing,samples were incubated at room temperature for 1 h with anti-digoxigenin antibody conjugated with peroxidase, washed withPBS, and developed with diaminobenzidine. Slides were observedin an Observer.Z1 fluorescence microscope equipped with a CCDcamera model AxioCAM MRM (Carl Zeiss, Inc., Thornwood, NJ).

3. Results and discussion

3.1. FC cDNA from R. prolixus was isolated, sequenced, and usedto build a sequence database

Clones (total of 494) were sequenced and used to assemblea database (see Supplemental Table S1) that, after clusterization,yielded 341 unique sequences (320 singletons and 21 contigs).Approximately 71% of all sequences showed matches with an e-value<10�5 to the NR protein database. All pertinent information wasincluded in anExcel spreadsheet (Supplemental Table S1) andused formanual annotation of sequences (included in column O). The anno-tated sequences were grouped into 15major functional classes (Fig.1;Supplemental Tables S1, column P and Table S2, column AM). Theoverall picture of the transcriptome showed a tissue highly committedto transcription, protein synthesis, and secretiondfunctions thataccounted formore than two-thirds of all transcripts. Only contigs thatgave a positive match with the R. prolixus genome were included inthis analysis (Assembly 1.0, available at http://genome.wustl.edu/tools/blast/Rhodnius_prolixus-1.0.1). The results of the BLAST of thecontigs against the genomecanbe found in columnQof SupplementalTable S1.

3.2. Secreted (S) proteins and genes possibly related to follicleand oocyte development

Sproteins represent a contributionof follicle cells to oocyte contentor to eggshell. Searching the open-reading frames for candidate Sproteins showing a signal peptidedand as a help to guide annota-tiondthe contigs from Supplemental Table S1 were six-frame trans-lated and the resulting peptides submitted to http://www.cbs.dtu.dk/services/SignalP/. The prediction results (Supplemental Table S1column K in Spreadsheet ALL) were manually examined, and those

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in which the initial methionine was present were transferred toSupplemental Table S1, spreadsheet SignalP. We identified eight con-tigs coding for S proteins (Supplemental Fig. 3) and another sevencoding formembrane proteins. Three of those contigs are composedofmultiple reads but have no reliable match in any available proteindatabase.

The most highly expressed message (contig 1, herein namedrpSPRP, for small proline-rich protein) accounted for about 8% of alltranscripts. This cDNA codes for a protein of 137 aa, which isexpected to be converted into a mature protein of 118 aa residuesand an estimate mass of 13.2 kDa. It has a low-complexity sequencein which the most conspicuous feature is the high proline content(21 residues; about 18%). Although similar proteins have not beendescribed in insects, in mammalian tissues, SPRPs were identifiedas important components of epithelial tissue (Nemes and Steinert,1999). SPRPs have been shown to take part in formation of the so-called cornified cell envelope, which is a hard layer made ofprotein/lipid polymer that stays on the exterior of dead cornifiedcells of the skin. Several proteins (including SPRPs) are covalentlylinked by protein crosslinking to form this layer, which is thought tobe important in conferring biomechanical properties to the corni-fied cell envelope that allow it to function as a protective barrier,which is the essence of the squamous epithelial tissue, a physio-logical role that is also performed by insect egg chorion. Analysis ofgene expression shows that rpSPRP expression is basically limitedto the epithelium of vitellogenic follicles (Fig. 2A). This could resultfrom either rpSPRP performing a role in yolk formation or beinga component of follicle ECM or, alternatively, could be explained byaccumulation of rpSPRP during vitellogenic growth followed byincorporation into eggshell only later, during choriogenesis.

Another putative S protein (contig 162) showed significanthomology to chitin-binding protein components of the insectcuticle that has a non-cysteine-based chitin-binding domain,thereby being a typical chorion protein candidate (SupplementalFig. 2B). It has been shown that chitin is an important componentof the insect eggshell (Moreira et al., 2007); therefore, chitin-binding proteins made by FE may also participate in formation ofchorion. This mRNA coded for a 125-aa precursor and a small 105-residue mature protein, which was about equally expressed byepithelia of both vitellogenic and choriogenic follicles (Fig. 2B),

Fig. 2. Expression of selected secreted proteins during developmental stages of ovarian follicor from follicles already engaged in chorion formation were dissected and mRNA levels omean � SEM (n ¼ 4).

reinforcing the point that some chorion proteins may be expressedbefore actual formation of the egg envelope. Contigs 232 and 296(Supplemental Tables S1 and S2) codify for proteins with a strongcandidate signal peptide (Supplemental Fig. S3) that shows a low-complexity amino acid profile with relatively high abundance ofglycine (26 and 20% of the amino acid residues of the matureprotein, respectively). Glycine-rich proteins are frequently foundamong components of ECM, being identified also as components ofthe cornified envelope of mammalian skin such as loricrin (Nemesand Steinert, 1999). Glycine-rich domains are also found in spiderfibroin, where repeats of GA or GS resembling those found incontigs 232 and 296 are thought to be involved in determination ofmechanical properties of silk web (Gosline et al., 2002, 1999).

A partial sequence for one of the secreted proteins (Rp45, contig97; Supplemental Table S1 and Supplemental Fig. 1) had alreadybeen reported, being described as a component of Rhodniuseggshell (Bouts et al., 2007). As expected for a major chorioncomponent, evaluation of expression of this message by qPCRshowed that this protein is exclusively expressed during chorio-genesis (Fig. 2C), confirming the developmental profile describedbefore (Bouts et al., 2007).

One mechanism that has been shown to produce hardening ofthe egg chorion is the oxidative crosslinking of proteins promotedby the action of peroxidases, such as the ovoperoxidases (Edenset al., 2001; Heinecke and Shapiro, 1990; Margaritis, 1985), whichneed a source of hydrogen peroxide to catalyze the formation ofdityrosine. This task could well be fulfilled by the extracellular SODcoded by contig 417 (Supplemental Tables S1 and S2).

Contig 531 codes for a putative S protein (Table S1, SupplementalFig. S3) that would fit in the class of odorant-binding proteins,a group that already has several members that do not bind odormolecules but other types of hydrophobic ligands, such as heme asthe Rhodnius heme-binding protein (Dansa-Petretski et al., 1995;Paiva-Silva et al., 2002) or biogenic amines such as the D7 familyof proteins found in the saliva of Diptera (Calvo et al., 2006). Oneinteresting candidate ligand for this protein would be juvenilehormone, as the Drosophila take-out proteindalso a member of theodorant-binding protein familydhas been shown to bind juvenilehormone andmodulate development of themale reproductive tract(Dauwalder et al., 2002; Noriega et al., 2006).

le Tropharium or follicular epithelium from follicles at pre-vitellogenesis, vitellogenesisf three transcripts showing signal peptides were evaluated by qPCR. Data shown are

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Contig 306 codes from a homolog of Jagunal (SupplementalTables S1 and S2), an evolutionarily conserved transmembrane ERprotein that is found in both oocytes and follicular cells of theDrosophila ovary, where it has been related to reorganization of theER during follicle development and ER-derived vesicle trafficking(Lee and Cooley, 2007). Jagunal comes from a Korean word thatmeans “small egg”, as mutations for this gene are lethal and wereshown to lead to abnormal follicle morphology.

During oogenesis, the FE is exposed tomechanical stretching dueboth to the large rate of growth of the vitellogenic oocyte and tocontraction of the ovarian sheath muscle. Ezrin/radixin/moesin(ERM) proteins (Supplemental Tables S1 and S2, contig 124) arelocated at the cell cortex and interact with actin filaments, bridgingthem with the plasma membrane, and play a major role in thisprocess. The dramatic changes in cell volume and shape that occurduring oocyte development and the opening of large spacesbetween epithelial cells are needed to expose the oocyte surface tohemolymph, thus allowing receptor-mediated uptake of vitelloge-nin, the yolk protein precursor that is 80% of the protein content ofmature Rhodnius eggs (Oliveira et al., 1986). This opening of inter-cellular spaces, termed patency, is known to be under the controlof juvenile hormone (Davey, 2007) and requires extensive rear-rangement of cytoskeleton and cell volume. ERM proteins(Supplemental Tables S1 and S2, contig 124) are also involvedin formation of microvilli, cellecell adhesion, cell shape, andmembrane trafficking and seem to be part of this process. In addi-tion to rearrangement of the cytoskeleton, these proteins integratediverse signaling pathways, are phosphorylated by protein kinases,are capable of binding phosphatidylinositol 4,5-bisphosphate

Fig. 3. Expression of selected genes related to reticulu

(PI(4,5)P2), and interact with small GTP-binding proteins (Niggliand Rossy, 2008).

A VASA-like transcript (Supplemental Tables S1 and S2, contig322) was found in this transcriptome and might play a role inoocyte development. VASA proteinsdDEAD-box RNA-bindingproteins with an RNA dependent helicase activity-are required forpole determination and completion of oogenesis in Drosophila(Abdelhaleem, 2005; Styhler et al., 1998; Tinker et al., 1998). InDrosophila, VASA expression takes place in the oocyte, germ cells,and nurse cells (Raz, 2000); however, in the lizard Podarcis sicula,VASA protein is localized also in a group of follicle cells of somaticorigin that perform functions that in Drosophila are characteristic ofnurse cells, transferring molecules and eventually their entirecytoplasm to the oocytes by means of intercellular bridges(Maurizii et al., 2009). Because cytoplasmic connections betweennurse cells and oocyte are broken during mid-vitellogenesis(Bjornsson and Huebner, 2004), it is interesting to speculate thatVASA may enable FE cells from the telotrophic ovary of hemip-terans to perform functions that in Drosophila are restricted tonurse cells.

3.3. Protein synthesis, secretion, and control of cell death

Global analysis of functional classification of transcripts (Fig. 1,Supplemental Tables S1 and S2) showed that close to two-thirds ofall proteins expressed that had a functional category (excludingproteins with unknown function) were somehow related to tran-scription, translation, protein turnover, and vesicular traffic,reflecting the immense metabolic effort accomplished by follicular

m stress during development of ovarian follicles.

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cells in providing their contribution to egg maturation (Fig. 1). Thispicture fits well with the abundant ER that occupies almost all thecytosol of binucleated FE cells (Atella et al., 2005). In the last fewyears, the concept of RS has been well established, defining a linkbetween intense protein synthesis/secretion and control of pro-grammed cell death. Proteins targeted for secretion enter the ER asunfolded polypeptides with reduced cysteines. Maturation of theseproteins in the secretory pathway involves oxidative formation ofthe disulphide bond and reshuffling; hence, proper protein foldingis highly dependent on fine tuning of redox equilibrium in the ER.The so-called unfolded-protein response (UPR) frequently observedduring cellular stress conditionsdincluding increased proteinsecretiondis intrinsically related to the mechanisms that controlcell death and survival and has been the subject of an extensiveliterature recently reviewed (Tabas and Ron, 2011). Several tran-scripts identified here are potentially involved in the control of celldeath (Supplemental Table S1, Supplemental Fig. S2) and could play

Fig. 4. Apoptotic cell death occurs at the end of chorion formation A, B, Trypan blue exclufollicle) and ch (choriogenic oocyte). Bars: 1 mm, 2 mm, respectively. C, D, Follicular epithelichoriogenic oocytes showing labeled nuclei (arrows) after TUNEL assay. Bar: 120 mm. G, H, Dbinucleated.

an important role in the preservation of FE cells facing a conditionof RS. Initially, we hypothesized that this would mean that controlof RS would be a major priority of follicular cells that were activelyengaged in secretion of oocyte yolk and chorion proteins. This ledus to evaluate expression of a selected set of genes by qPCR.Measurement of the expression of six transcripts probably involvedin the control of UPR and prevention of programmed cell deathshowed expression to be increased during early stages of oogenesisand repressed at late stages, especially in the choriogenic follicle(Fig. 3; defender against cell death [DAD]-like, HSP90, DNAJ/HSP40,Sec24, Sec61, and 14-3-33). The Dad gene (contig 259, Fig. 3A,Supplemental Fig. S2, panel D) (Kelleher and Gilmore, 1997) isa subunit of an oligosaccharyltransferase, and mutants with trun-cated forms of DAD were shown to present increased apoptosis(Nakashima et al., 1993), while higher expression led to survival(Sugimoto et al., 1995), reflecting the importance of N-glycosylationfor protein traffic in the EReGolgi pathway and the link between ER

sion assay. Arrows indicate regions with dead cells. tr (tropharium), Vg (vitellogenicum of vitellogenic oocytes after TUNEL assay. Bar: 120 mm. E, F, Follicular epithelium ofetail of the TUNEL label in different nuclei. Bar: 50 mm. Note that the epithelial cells are

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stress and cell death. In a similar way, Sec24 (contig 73, Fig. 3F andSupplemental Fig. S2, panel B) is an essential component of the coatprotein complex (COPII) that works in secretory vesicle formationand is involved in selection of newly synthesized proteins forexport (Pagano et al., 1999; Peng et al., 2000). Sec61 (contig 46,Fig. 3E, Supplemental Fig. S2, panel A) also participates in assemblyof the secretory highway in the Sec61 complex that constitutesa channel for signal peptide-dependent protein import and retro-grade transport of misfolded proteins out of the ER (Robson andCollinson, 2006). Chaperones of the Hsp90 family (contig 488,Fig. 2E) and the DNAJ/Hsp40 (a co-chaperone of HSP70) (contig 516,Supplemental Fig. S2, panel F) are also well known for theirparticipation in protein folding and secretion (Qiu et al., 2006). TheHsp70/DNAJ chaperone has been shown to act in UPR, preventingapoptosis induced by CHOP (Gotoh et al., 2004). 14-3-33 proteins(contig 446, Supplemental Fig. S2, panel C) homologs are down-regulated at late oogenesis (Fig. 3D). Proteins of this class have beenimplicated in several signal transducing pathways (Fu et al., 2000)including cell-cycle control in Drosophila (Su et al., 2001) anddefinition of oocyte and epithelial cell polarity (Benton and StJohnston, 2003), although their precise role in cell death controland survival is less well defined concerningmolecular mechanisms.Evidence relating 14-3-33 proteins to apoptosis show that theseproteins are capable of associating with BAD and that cleavage bycaspase 3 releases BAD, which in turn associates with Bcl-2 (Qi andMartinez, 2003).

Inmost eukaryotic cells, the expected response of being exposedto conditions that induce UPR is increased expression of survivaltranscripts, as observed in the fungi Aspergillus nidans (Guillemetteet al., 2007). Lee et al. (2003) have described a similar set of genes,the transcription of which is induced by XBP-1 and that are thoughtto be the chaperone effectors of the UPR in the ER; however, weshow here that FE at late developmental stages not only does notincrease expression of this group of genes but rather showsa pronounced trend to lower its transcription (Fig. 3).

In contrast to the more common pattern of cellular response oftriggering the UPR gene set to preserve cell integrity, these resultsshow downregulation of UPR-related genes when there is intenseER traffic. After follicle cells complete chorion secretion, theydegenerate and die, forming a residual body, referred to by someauthors as corpus luteum (Baum et al., 2005; Wigglesworth, 1953).Therefore, programmed death of follicle cells should be taken aspart of the oogenesis developmental program. In accordance withthis (Nezis et al., 2006, 2002) reported the occurrence of pro-grammed cell death in degeneration of ovarian follicle cells ofhigher Diptera. Therefore, herewe propose as aworking hypothesisthat death of cells in the ovarian follicle is fueled by the RS owing toits effort to synthesize chorion proteins. In line with this hypoth-esis, we found evidence that clusters of cells from the FE die in theend of oogenesis, as showed by Trypan blue staining exclusionassay in choriogenic oocytes (Fig. 4A and B, arrows). Additionally, itwas possible to detect TUNEL-positive cells randomly dispersed inthe epithelium of choriogenic oocytes (Fig. 4EeH, arrows) (1.5� 0.9TUNEL-positive cells/400 mm2) and not in the epithelium of vitel-logenic oocytes (Fig. 4C and D). The process of cell death seems tobe very rapid and is apparently triggered by local interactionsbetween cells, as Trypan blue-stained cells were always found asclusters and TUNEL-positive cells were only found in the epithe-lium of oocytes at late choriogenesis. It was described in Drosophilathat follicle cells are interconnected with intercellular bridges (gap-junction type) that are confined to arrays of no more than eightcells, and that several of these independent clusters are found inthe epithelium (Woodruff and Tilney, 1998) (Haglund et al., 2010).The authors discuss that cell-to-cell movement throughout theepithelium of global cytosolic regulatory molecules cannot occur

via these intercellular bridges, but weak signals affecting only oneor a few cells in each cluster would be amplified by spread throughthe intercellular bridges. Thus, we cannot rule out the possibilitythat the signaling events that trigger cell death at late oogenesisin R. prolixus may be associated with these types of intimateconnections between the follicle cells, resulting in the clusterpatterning that we observed by Trypan blue staining.

4. Conclusions

The field of developmental biology is heavily biased towardembryogenesis. Egg components that are synthesized and properlyassembled during oogenesis constitute the starting point forgrowth of a viable embryo. Here we present evidence that inR. prolixus at late stages of egg formation, programmed death offollicle cells occurs and we propose that this simultaneity is notmerely a fortuitous coincidence. Instead, we propose that the deathof follicle cells is a crucial part of the oogenesis developmentalprogram and occurs to help in building of the chorion, a protectivecoat that will increase the biologic success of insect progeny. Ourdata show that downregulation of expression of cell survival-inducing transcripts occurs simultaneously with the increasedexpression of secreted proteins. Although we do not have evidenceto prove a causeeconsequence relationship between these twoevents, their simultaneity leads us to hypothesize that develop-ment of endoplasmic RS is the driving force for FE cell death, whichmarks the termination of oogenesis. In this case, FE cell survivalwould not serve to increase the biologic success of this organism.On the other hand, death and degeneration of these cells fit thesurvival program of this species’ offspring. This unconventionalregulation of UPR in ovarian cells is an interesting model forresearch on the relation between RS and cell death control.

Acknowledgements

We thank S.R. Cássia for technical assistance and NIAID intra-mural editor B. R. Marshall. This workwas supported by grants fromConselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq), Coordenação de Aperfeiçoamento de Pessoal de NívelSuperior (CAPES), Fundação de Amparo a Pesquisa de Estado do Riode Janeiro (FAPERJ), Fundação Universitária José Bonifácio (FUJB),INCT-Entomologia Molecular, and Howard Hughes Medical Insti-tute (HHMI, to PLO). JMCR was supported by the IntramuralResearch Program of the Division of Intramural Research, NationalInstitute of Allergy and Infectious Diseases, National Institutes ofHealth.

Because JMCR is a government employees and this is a govern-ment work, the work is in the public domain in the United States.Notwithstanding any other agreements, the NIH reserves the rightto provide the work to PubMedCentral for display and use by thepublic, and PubMedCentral may tag or modify the work consistentwith its customary practices. You can establish rights outside of theU.S. subject to a government use license.

Appendix. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ibmb.2011.06.004.

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