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Steroids 76 (2011) 1149– 1159
Contents lists available at ScienceDirect
Steroids
jo ur nal homep a ge: www.elsev ier .com/ locate /s tero ids
helminth cestode parasite express an estrogen-binding protein resembling alassic nuclear estrogen receptor
lizabeth Guadalupe Ibarra-Coronadoa, Galileo Escobedob, Karen Nava-Castroc,hávez-Rios Jesús Ramsesa, Romel Hernández-Belloa, Martìn García-Varelad, Javier R. Ambrosioe,livia Reynoso-Ducoinge, Rocío Fonseca-Linánf, Guadalupe Ortega-Pierres f, Lenin Pavóng,aría Eugenia Hernándezg, Jorge Morales-Montora,∗
Departamento de Inmunología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, AP 70228, México D.F. 04510, MéxicoUnidad de Medicina Experimental, Hospital General de México, México D.F. 06726, MéxicoDepartamento de Infectología e Inmunología, Instituto Nacional de Perinatología, México D.F. 11000, MéxicoDepartamento de Zoología, Instituto de Biología de la Universidad Nacional Autónoma de México, México D.F. 04510, MéxicoDepartamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Edificio A, 2do piso, Ciudad Universitaria, México D.F. 04510,éxico
Departamento de Genética y Biología Molecular, Cinvestav IPN, Av. Instituto Politécnico, Nacional 2508 Col. San Pedro Zacatenco 07360, MexicoDepartamento de Psicoinmunología, Instituto Nacional de Psiquiatría “Ramón de la Fuente”, México D.F., México
r t i c l e i n f o
rticle history:eceived 25 January 2011eceived in revised form 7 April 2011ccepted 10 May 2011vailable online 19 May 2011
eywords:uclear estrogen receptorstradiolaenia crassicepsysticercusarasiteormone receptors
a b s t r a c t
The role of an estrogen-binding protein similar to a known mammalian estrogen receptor (ER) is describedin the estradiol-dependent reproduction of the helminth parasite Taenia crassiceps. Previous results haveshown that 17-�-estradiol induces a concentration-dependent increase in bud number of in vitro cul-tured cysticerci. This effect is inhibited when parasites are also incubated in the presence of an ERbinding-inhibitor (tamoxifen). RT-PCR assays using specific oligonucleotides of the most conserved ERsequences, showed expression by the parasite of a mRNA band of molecular weight and sequence cor-responding to an ER. Western blot assays revealed reactivity with a 66 kDa protein corresponding to theparasite ER protein. Tamoxifen treatment strongly reduced the production of the T. crassiceps ER-like pro-tein. Antibody specificity was demonstrated by immunoprecipitating the total parasite protein extractwith anti-ER-antibodies. Cross-contamination by host cells was discarded by flow cytometry analysis.ER was specifically detected on cells expressing paramyosin, a specific helminth cell marker. Parasitecells expressing the ER-like protein were located by confocal microscopy in the subtegumental tissueexclusively. Analysis of the ER-like protein by bidimensional electrophoresis and immunoblot identified
a specific protein of molecular weight and isoelectric point similar to a vertebrates ER. Sequencing of thespot produced a small fragment of protein similar to the mammalian nuclear ER. Together these resultsshow that T. crassiceps expresses an ER-like protein which activates the budding of T. crassiceps cysticerciin vitro. To the best of our knowledge, this is the first report of an ER-like protein in parasites. This find-ing may have strong implications in the fields of host-parasite co-evolution as well as in sex-associatedction,
susceptibility to this infe. Introduction
The sex steroid hormone 17-�-estradiol (E2) acts upon theeproductive system of mammalians by binding to specific estro-
en receptors (ER), which determines changes in reproductivehysiology and behaviour [1,2]. Estrogens also transiently inducenumber of nuclear proto-oncogenes, such as the c-fos and c-jun
∗ Corresponding author. Tel.: +52 55 56223158; fax: +52 55 56223369.E-mail addresses: [email protected], [email protected]
J. Morales-Montor).
039-128X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.steroids.2011.05.003
and could be an important target for the design of new drugs.© 2011 Elsevier Inc. All rights reserved.
family proteins, which act as transcription factors through theER system in the endometrial epithelium of mature and imma-ture rodents. Changes in concentrations of these gene productspresumably trigger the proliferation and differentiation of theuterine epithelium and mediate effects in areas of the brain underhormonal control [3,4]. Recently, estrogens, and particularly 17-�-estradiol, have been shown to participate not only in reproductivephysiology, but in a number of different functions, including
immune modulation, brain activity, bone metabolism, lung andheart physiology. Also, sex steroids influence a wide array offunctions related to reproduction as well as to non-reproductivebehaviours. The broad distribution, age, sex and tissue-depending
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xpression pattern of estrogen receptors, as well as the functionalisruptions in receptor knockout animals are solid evidence of thereat diversity of the complex estrogen–estrogen receptor actions.
ER operates as a hormone-induced transcription factor whichrompts sexual receptivity in rats, hamsters, guinea pigs and mice5]. The ER may be expressed either as ER-� or ER-� [6–8]; theseariants are located in two different genes, and also differ function-lly depending on the tissue in which they are expressed [6,9,10].urthermore, estrogens affect the pattern of immune responsegainst different pathogens including parasites [11].
Recent experimental evidence suggests that steroid hormonesave direct effects on different stages of both helminthes, Tae-ia crassiceps and Taenia solium [12–15]. Specifically, androgenseduce the reproduction and viability of T. crassiceps metacestodesn a concentration-dependent manner [12,13] while 17-�-estradiolnd progesterone stimulate the proliferation rate in this parasite12].
Interestingly, tamoxifen (an anti-estrogen widely used in thereatment of estradiol-dependent breast cancers) exerts a strongoxic effect upon T. crassiceps, decreasing parasite reproduction initro and parasite loads in vivo [16]. Also, it has been shown that pro-esterone increases T. solium scolex evagination and worm growthn a concentration-independent way, while RU486, a progesteronentagonist, inhibits either scolex evagination or worm develop-ent induced by progesterone [14]. Despite of the fact that there
s a clear direct effect of steroid hormones on some parasites, theechanisms involved have not been fully defined yet. Onchocerca
olvulus has a nuclear receptor potentially able to bind retinoic acid17]. Furthermore, sequences related to a progesterone receptorere detected by RT-PCR and Western blot in the helminth par-
site T. solium [14]. Moreover, the presence of a possible mRNAequence similar to an estrogen receptor has been shown in T.rassiceps cysticerci [12] by RT-PCR and by sequencing a specificmplified fragment.
The present study was designed to search for an estrogeneceptor-like molecule, which could mediate the proliferativeffects of exogenous and endogenous 17-�-estradiol on theelminth parasite T. crassiceps. Results showed that T. crassiceps
ndeed has a protein similar in function to an ER, which plays a rolen parasite development. These findings may improve our under-tanding of the host-parasite molecular cross-talk, and could alsoepresent a target for the design of new drugs specifically directedo arrest the activity of key parasite molecules, such as transductionroteins and transcription factors involved in their establish-ent, growth and reproduction inside an immunocompetent
ost.
. Experimental
.1. Parasites
Cysticerci were obtained from intraperitoneally infected micend placed in tubes containing sterile PBS (1X) supplemented with00 U/ml of antibiotics-fungizone (Gibco, Grand Island). The tubesere centrifuged for 10 min at 200 × g at 4 ◦C, and the supernatantas discarded. Packed cysticerci were incubated in DMEM serum-
ree medium (Gibco 12491). The parasites were then centrifuged times at 200 × g for washing. After the final wash, viable cys-icerci (complete, translucent and motile cystic structures) wereounted under a stereoscopic microscope. Ten viable non-buddingysticerci of approximately 2 mm in diameter were then selected
nd dispensed into each well of 24-well culture plates (Falcon, Bec-on Dickinson Labware, Franklin Lakes, New Jersey) in 1 ml DMEMedium (Gibco 12491) and incubated at 37 ◦C and 5% CO2. A suffi-ient number of culture wells was prepared to evaluate the effects
oids 76 (2011) 1149– 1159
of in vitro treatment of estradiol and tamoxifen on cysticerci. Cul-tures were checked every day and their medium was completelyreplaced each 24 h or when it turned yellowish.
2.2. Ethics statement
Animal care and experimentation practices at the Instituto deInvestigaciones Biomédicas are frequently evaluated by the Insti-tute’s Animal Care and Use Committee, and by governmentalagencies, in strict accordance with the recommendations set forthin the Guide for the Care and Use of Laboratory Animals of theNational Institutes of Health of the USA, to ensure compliance withestablished international regulations and guidelines. The protocolwas approved by the Ethics Committee for Animal Experimentsof the Instituto de Investigaciones Biomédicas (Permit Number:2009-16). Mice sacrifice to obtain control tissues was performedunder sodium pentobarbital anesthesia, and all efforts were madeto minimize suffering.
2.3. In vitro treatment effects of E2 and tamoxifen on T. crassicepscysticerci reproduction
Culture grade 17-�-estradiol (E2) and tamoxifen were obtainedfrom Sigma. For in vitro tests, water-soluble E2 was dissolvedin DMEM serum-free culture medium, while tamoxifen was dis-solved in absolute ethanol. Next they were each prepared at aconcentration of 10 mg/ml and then sterilized by passage through0.2 mm Millipore filter paper. Each of the following experimentalconditions was applied to 24 parasite-loaded wells to obtain theconcentration-response curves: (a) parasites were supplementedwith serum-free DMEM as vehicle (control groups), (b) parasiteswere separately supplemented with 5, 10, 20 and 40 �g/ml ofE2 and (c) parasites were supplemented with 1, 10, 15, 40 and80 �g of tamoxifen. Optimal concentrations of E2 and tamoxifenwere selected from concentration-response curves, and later usedfor time-response curves. Final concentrations were: 40 �g/mlmedium of E2 and 40 �g/ml medium of tamoxifen. The numberof buds per cysticercus as a function of days elapsed in culture wasassessed as the variable response. Tamoxifen was supplemented2 h before the addition of 17-�-estradiol. Parasite reproduction wasmeasured by counting the total number of buds in the 10 cysticerciin each well. Bud count, as well as viability, was checked every dayunder an inverted light microscope (Olympus, MO21, Tokyo, Japan)at 4× and 10× magnification. Injury to cysticerci was recognizedmicroscopically by progressive internal disorganization, develop-ment of whitish opaque areas on the parasite’s tegument and lossof motility. Dead cysticerci were immobile, opaque and structurallydisorganized.
2.4. Detection of ER-like gene expression in T. crassiceps byRT-PCR
Total RNA was isolated from untreated, E2- and tamoxifen-treated T. crassiceps cysticerci, and from BALB/c AnN femalemouse uterus as control for specific ER gene amplifica-tion by the single-step method based on guanidine isothio-cyanate/phenol/chloroform extraction using Trizol reagent (Invit-rogen, Carlsbad, CA). Briefly, cysticerci were disrupted in Trizol(1 ml/0.1 g tissue), and 0.2 ml chloroform were added per 1 ml ofTrizol. The aqueous phase was recovered after 15 min centrifuga-tion at 16,000 × g. RNA was precipitated with isopropyl alcohol,washed with 75% ethanol and dissolved in RNAse-free water.
RNA concentration was determined by absorbance at 260 nmand its purity was verified by electrophoresis in 1.0% denatur-ing agarose gel in the presence of 2.2 M formaldehyde. Total RNAwas reverse-transcribed followed by specific PCR amplification of
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he ER-like gene from parasite tissue. The ribosomal 18S geneas used as control, as described elsewhere [18]. Briefly, 10 �g
f total RNA were incubated at 37 ◦C with 40 units of M-MLVeverse transcriptase (Applied Biosystems, USA) in 20 �l of reac-ion volume containing 50 �M of each dNTP and 0.05 �g oligodT) primer (Gibco, Invitrogen, NY). Ten microlitres of the cDNAeaction were subjected to PCR in order to amplify the sequencesf the specific genes. Primer design was based on the most con-erved regions of sequenced genes of all species reported in theatabase. Sequences of primers were as follows: ER-� (239 bp)ense 5′-AGACTGTCCAGCAGTAACGAG-3′ and the antisense primerequence 5′-TCGTAACACTTGCGCAGCCG-3′. The 50 �l PCR reactionncluded 10 �l of previously synthesized cDNA, 5 �l of 10× PCR-uffer (Perkin-Elmer, USA), 1 mM MgCl, 0.2 mM of each dNTP,.05 �M of each primer, and 2.5 units of Taq DNA (Biotecnologiasniversitarias, Mexico). After an initial denaturing step at 95 ◦C for
min, temperature cycling was as follows: 95 ◦C for 30 s, 57 ◦C for5 s and 72 ◦C for 45 s during 35 cycles. A further extension stepas completed at 72 ◦C for 10 min for each gene. The 20 �l of the
CR reaction were electrophoresed on 2% agarose gel in the pres-nce of a 100 bp ladder as molecular weight marker (Gibco, BRL,Y). The PCR products obtained were visualized by staining withthidium bromide. In both cases, different PCR conditions weressessed until a single band corresponding to the expected molec-lar weight of the gene was found. The ribosomal 18S gene is aonstitutively expressed gene and it was used as internal loadingontrol. The sequence of the house-keeping gene was as follows:8S (238 bp) sense primer 5′-GGGTCAGAAGGATTCCTATG-3′, andntisense primer 5′-GGTCTCAAACATGATCTGGG-3′.
.5. ER-like protein detection in T. crassiceps by Western blot
Total protein was obtained from T. crassiceps cysticerci byonventional Tris–HCl isolation. Briefly, non-treated, E2 andamoxifen-treated cysticerci were disrupted in Tris–HCl (1 ml/0.1 gissue) and proteases inhibitor cocktail (Calbiochem). Mouse uterusas used as internal control of protein extraction and integrity.
he supernatant was recovered after 15 min centrifugation at6,000 × g and the pellet was discarded. Protein quantity was deter-ined by absorbance at 595 nm using the Bradford-Lowry method.
hirty micrograms of total protein extracts of T. crassiceps cys-icerci and mouse uterus were boiled in reducing Laemmli sampleuffer, separated by SDS-PAGE (10% acrylamide) and transferrednto PVDF membranes. The membranes were blocked overnightn PBS buffer (0.2% Tween 20) containing 1% BSA. Then, different
embranes were washed 5 times in PBS-Tween and separatelyncubated for 1 h in presence of rabbit anti-ER-� (1:300, Santaruz Biotechnology). After this first incubation, membranes wereashed 3 times in PBS-Tween and subsequently incubated for 1 h
n presence of goat anti-rabbit IgG-HRP (1:500, Santa Cruz Biotech-ology Amersham) as secondary antibody. Immediately after theands were visualized the reaction was stopped using the ECL sys-em according to the manufacturer’s instructions (Super Signal ECL,ierce). Chemiluminescent signals were captured on Kodak Bio-ax film.
.6. Immunoprecipitation and specific detection of ER-likerotein in Taenia crassiceps
For specific detection of ER-like protein in T. crassiceps, 30 �gf total protein from the parasite were mixed with 1 �g of rabbitnti-ER-� during 1 h at 4 ◦C. Then 30 �g of Protein G was added
nd the mixture was incubated overnight at 4 ◦C. After centrifuga-ion at 5,000 × g for 1 min, the conjugate was washed 3 times with00 �l lysis buffer (Tris–HCl and proteases inhibitor cocktail) andentrifuged each time at 5000 × g for 1 min at room temperature.oids 76 (2011) 1149– 1159 1151
Once the supernatant was discarded, the pellet was resuspendedin 30 �l lysis buffer and 30 �l of Laemmli loading buffer.
2.7. Specific detection of ER-like protein in T. crassiceps cysticerciby flow cytometry
T. crassiceps cells were extracted by tissue disruption from cul-tured treated and non-treated parasites. Mouse spleen cells wereused as FACS calibration control. For each treatment, 2 × 106 cellswere incubated at 4 ◦C for 20 min in presence of anti-CD3 and anti-MHC-II or anti-CD4, anti-CD8 and subsequently washed in sterilePBS 1X-staining. Next, cells were centrifuged at 300 × g for 5 min,and incubated in GolgiPlug for 3 h. Cells were then washed inPerm/Wash buffer and centrifuged at 11,000 × g for 5 min. Afterthis, cells were separately incubated in presence of rabbit �-ER1 �g/�l (Santa Cruz, Biotech) and mouse �-paramyosin 1 �g/�l atroom temperature for 20 min, and subsequently washed in PBS 1X-staining. After this step, cells were centrifuged at 300 × g for 5 min.Cell pellets were resuspended separately in presence of the sec-ondary antibody FITC-conjugated goat anti-rabbit or PE-conjugatedrat anti-mouse antibody, and incubated at 4 ◦C for 30 min in thedark. After this second incubation, cells were washed in PBS 1X-staining solution and centrifuged at 300 × g for 5 min. Cell pelletswere resuspended in 500 �l of PBS 1X-staining solution in absenceof light and analyzed by flow cytometry using a FACS Calibur (BD,Biosciences). Data were analyzed with the FlowJo software.
2.8. ER-like protein location on T. crassiceps cells byimmunofluorescence
Cultured T. crassiceps cysticerci were washed with PBS 1X,embedded in Tissue Tek (Triangle Biomedical Science), and frozenat −80 ◦C. Parasite tissue sections (5 �m) were fixed with 4%paraformaldehyde for 30 min, washed 3 times in PBS and blockedfor 30 min with RPMI medium containing 0.5% albumin bovine and5% fetal bovine serum [19]. Cross-sections were then incubatedwith a 1:500 dilution of rabbit anti-ER-� (Santa Cruz, Biotech)for 45 min at 37 ◦C, washed with PBS and then incubated withfluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit anti-body (Zymed) at 1:200 dilution. Control experiments were assessedincubating the 5 �m thick tissue sections in presence of only theFITC-conjugated goat anti-rabbit antibody at the same dilution.To eliminate background fluorescence, samples were contrastedwith 0.025% Evans Blue for 10 min. After two single washings,samples were mounted in Vectashield mounting medium (VectorLaboratories Inc.) and examined with a Carl Zeiss epifluorescencemicroscope at 10× and 40× magnification (Carl Zeiss, Germany).
2.9. Cell sorting of T. crassiceps cells
For cell sorting, cells were obtained as previously stated, andwere first selected by size and granularity into 4 subpopulationsof parasitic cells, and electronically purified using a FACSAria cellsorter (Becton & Dickinson). Cells were recovered into an Eppendorftube containing 5% of culture medium supplemented with bovinefetal serum. Cells were then used to perform binding experiments.Purity of the sorted populations was above 96%.
2.10. Preparation of cysticercus proteins
After recovering the parasites, these were sonicated 3 timeswith a period of amplitude frequency of 50 kHz and 1 min inter-
vals between each sonication. Protein purification was performedafter its precipitation at 4 ◦C using a −20 ◦C frozen acetone solu-tion containing 10% TCA/20 mM DTT. Precipitated proteins wereresuspended in a commercial 5 mM Tris-Base complemented with
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nhibitors (pepsin 1 mg/ml, pepstatin 2 �g/ml, aprotinin 5 �g,eupeptin 5 �g/ml, PMSF 1 mM) or in 6.7 mM phosphate bufferdjusted to pH 7.4 with 0.04 M KCl and 1 mM MgCl2 in presence ofroteases inhibitor. Resultant proteins were quantified using theC Protein Assay commercial kit and bovine serum albumin as a
tandard. Protein aliquots were frozen at −70 ◦C until used.
.11. Electrophoresis
Ten percent SDS-PAGE gels were run for protein stacking at0 V during 20 min, and electrophoretic conditions were 100 V dur-
ng 90 min. After separation, proteins were revealed by Coomassielue staining (PhastGel Blue R). The standard commercial molecu-
ar weight marker was kaleidoscope (Bio-Rad). Gels were unstainedsing a solution containing glacial acetic acid, methanol andater. Gel images were captured with an XRS ChemiDoc (Bio-Rad)hotodocumentation device equipped with Quantity One softwaredkdkd.
.12. Isoelectrofocusing
IPG strips (GE) of 7 cm length (pH 3–10) were hydrated at roomemperature for 14 h in the presence of protein (50 �g) and a rehy-ratation solution containing 7 M urea, 2 M thiourea, 4% CHAPS,–10 2% ampholytes, 60 mM DTT and 0.002% bromophenol blue.roteins were separated by pI in a Protean IEF Cell equipmentBio-Rad) at 20 ◦C using 50 �A/strip in a discontinuous current asollows: step 1 with a linear slope during 20 min at 250 V, step 2ith a linear slope during 2 h at 4000 V and step 3 with a rapid steep
lope beginning at 4000 V and ending at 10,000 V. After finishing,trips were recovered and frozen at −70 ◦C until used.
.13. Second dimension electrophoresis
After isoelectrofocusing, strips were molded with 0.5% hotgarose and 2D electrophoresis was performed at 4 ◦C using a com-ercial system (NuPAGE® Bis–Tris electrophoresis system) with
re-cast 4–12% mini gels at 200 V during 40 min. During runnings,uPAGE® antioxidant reagent for reducing conditions was used. All
eagents were from Invitrogen. Gels were stained with Sypro-RubyGE, Healthcare) or Blue Coomassie following the suppliers’ indi-ations. Other 2D gels were processed for protein electrotransfero PVDF membranes as indicated below. Gel images were cap-ured with the PDQuest software (BioRad). Exact pI values werealculated after printing the images and tracing the MW vs. pIoordinates, according with standard MW markers. The apparentW/pI parameters of each resultant spot was estimated. Selected
pots were cut out from gels in 18.2 m� MilliQ water and main-ained at 4 ◦C until further processing.
.14. Protein electrotransference
Proteins on SDS gels were transferred, under reducing condi-ions, to PVDF membranes (GE, Healthcare) using a commercialquipment XCell II Blot Module coupled to a chamber XCellureLockTM Electrophoresis Cell and Novex Mini-Cell in presence ofuffer (NuPAGE® Transfer Buffer). All reagents were obtained fromnvitrogen.
.15. ER-like protein detection by 2D electrophoresis andestern blot
Briefly, after processing proteins and separating them by 2D gellectrophoresis, they were transferred into nitrocellulose mem-ranes and Western blot was performed using an anti-ER-�ntibody, as mentioned before. Only the immunodetected point,
oids 76 (2011) 1149– 1159
corresponding to the expected molecular weight and to the pre-dicted isoelectrical point is highlighted in the blotted membrane.
2.16. Phylogenetic analysis of the putative T. crassiceps estrogenbinding protein
The protein sequence of the T. crassiceps estrogen binding pro-tein was aligned to the ER-� protein sequences of other 13 species(including mammals, birds, fish, one reptilian and one amphibian)obtained from protein data sets in GenBank. Sequence alignmentwas done using Clustal W software. Alignment of contained 131amino acids (aa) from 13 different taxa. Phylogenetic relationshipswere inferred using the Neighbor joining (NJ) method. Robustnessof the NJ tree was evaluated using bootstrap of 10,000 replicates.The tree was drawn using RETREE and DRAWGRAM from PHYLIP.The genetic differentiation between taxa was estimated using themean character difference with the help of PAUP* 4.0b10 software.It is important to point out that the number of species used forthe analysis was selected based on the ER-� sequence found in theGene Data Bank (for some species there is only one sequence).
2.17. Experimental design and statistical analysis
E2 and tamoxifen concentration-response and time-responsecurves were estimated from six independent experiments; eachwas performed with 10 cysticerci, freshly extracted from differentinfected donor female mice. Each experiment was replicated in 24different wells. The response variable used in statistical analysiswas the sum of buds present in the 24 wells with each treatmentand time of exposure of the experiments. Data from the six replica-tions of each experiment were pooled and expressed as mean ± SD.All optical densitometries as well as mean fluorescence of the flowcytometry analysis were calculated for 4 different experiments,and expressed as mean ± SD. Data were analyzed using either Stu-dent’s t-test or one-way ANOVA and subsequently with Dunnet’sMultiple Comparison Test, depending on the experimental design.Differences were considered statistically significant with P < 0.05.
3. Results
3.1. E2 stimulates while tamoxifen diminishes T. crassicepsreproduction
Bud number of cultured T. crassiceps cysticerci clearly increasedupon addition of 17-�-estradiol, in a concentration-dependentmanner. Compared to control groups, E2 increased parasite repro-duction rate 2-fold at the lowest concentration (0.1 �g/ml), andmore than 10-fold at the highest concentration (40 �g/ml), withoutaffecting parasite viability (Table 1). In addition to the concentra-tion effects, the proliferative action of E2 on parasite reproductionwas maintained throughout the approximately five days of in vitroculture (Table 1). In contrast, tamoxifen showed the opposite effecton bud number, although this effect was only significant with thehighest drug concentrations (20 and 40 �g/ml). This suggests thatthe tamoxifen effect was also dependent on drug concentration(Table 2). Interestingly, when compared with control cysticerci,tamoxifen treatment of the larvae had no significant effects onparasite basal reproductive rate (Table 2). However, it is clear thattamoxifen completely blocked the E2 dependent proliferative effect(Table 2), emphasizing the importance of the E2-dependent prolif-erative mechanisms in T. crassiceps cysticercus reproduction.
3.2. ER-like mRNA gene expression in Taenia crassiceps
Using specific primers, which were designed considering themost conserved sequences of all ER reported to date, we were able
E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149– 1159 1153
Table 1Dose-response curve reproduction of cultured Taenia crassiceps cysticerci inresponse to estradiol.
Estradiol concentration (�g/ml) Total number of buds Cysticerci motility
0 10 ± 1.123 ***0.1 24 ± 9.8944 ****1 36 ± 11.0954 ****
10 73 ± 20.7527 *****20 96 ± 23.8164 *****40 123 ± 25.7527 *****
Data represent mean ± SD number of buds from 3 experiments, with 10 cysticerciper well, 6 wells per dose, during five days of culture.* Refers to a scale used by us to describe the motility of the organism. Injury tocysticerci was recognized microscopically by progressive internal disorganization,development of whitish opaque areas on the parasite’s tegument, and by loss ofmotility. Dead cysticerci were immobile, opaque, and disorganized structures. (***)50% of cysticerci motile, but 100% alive, (****) 80% of cysticerci motile, but 100% alive,(*****) very motile, 100% alive and healthy in their appearance.
Table 2Dose-response curve of tamoxifen effect on estradiol-stimulated reproduction ofcultured T. crassiceps cysticerci.
Tamoxifen (�g/ml)/estradiol(�g/ml) concentration
Total number of buds Cysticerci motility
0/0 10 ± 1.123 *****0/1 24 ± 0.8944 *****5/10 16 ± 1.0954 ****
10/15 13 ± 0.7527 ***20/40 6 ± 0.8164 **40/80 3 ± 0.7527 *
Data represent mean ± SD from 3 experiments, with 10 cysticerci per well, 6 wellsper dose.* Refers to a scale used by us to describe the motility of the organism. Injury tocysticerci was recognized microscopically by progressive internal disorganization,development of whitish opaque areas on the parasite’s tegument, and by loss ofmotility. Dead cysticerci were immobile, opaque, and disorganized structures. (*****)Very motile, and healthy in their appearance, (****) less than 80% of cysticerci motile,bn1
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Fig. 1. Expression of an estrogen receptor-like protein in Taenia crassiceps. (A) Asingle band of approximately 239 bp corresponding to ER was detected in all in vitro17-�-estradiol-treated cysticerci. Tamoxifen treatment significantly inhibited theER -like protein expression compared to control cysticerci. (B) Relative expressionof the T. crassiceps ER-like gene using 18S rRNA as constitutive gene. E2, cysticercitreated with 17-�-estradiol; Tmx, cysticerci treated with tamoxifen; Tmx + E2, cys-
ut still 100% alive, (***) less than 50% of cysticerci motile, but still 100% alive, (**)o motility in 80% of the cysts, with a reduction of 50% in survival, (*) no motility in00% of the cysts, with 100% mortality reached.
o amplify a band of 239 bp corresponding to the expected length ofhe T. crassiceps ER-like gene (Fig. 1A) and to the ER gene of mouseterus used as internal control. Interestingly, no difference in ER-
ike gene expression was observed between E2-treated and controlarasites. However, tamoxifen-treated cysticerci showed a signifi-ant down-regulation of T. crassiceps ER-like gene expression, evenn presence of E2 (Fig. 1B).
.3. ER-like protein expression by Western blot andmmunoprecipitation in T. crassiceps
Native ER-like protein was detected in untreated, and in E2nd tamoxifen-treated parasites by the Western-blot method. TheR-like protein of T. crassiceps was detected, in addition to somenspecific bands (Fig. 2A). Immunoprecipitation was then used tobtain the specific ER-like protein from the parasite. Contrary to theata obtained by RT-PCR, no changes in total protein quantity werebserved with any treatment and the ER-like protein was detectedn all treatments (Fig. 2B).
.4. ER-like protein is specifically detected in T. crassiceps and its not a contamination product from host immune cells
Flow cytometry analysis first showed that T. crassiceps cells
iffered in size and complexity from mouse spleen cells. In fact,arasite cells were approximately 3-fold smaller and showed lessomplexity (Fig. 3A) than mouse spleen cells (Fig. 3B). In addi-ion, parasite cells showed no expression of the membrane markersticerci treated with tamoxifen plus 17-�-estradiol; C, cysticercus culture withouttreatment; CEtOH, cysticercus culture with vehicle; uterus, mouse uterus. *P < 0.05,**P < 0.01.
CD4+ and CD8+ which are typically present in some types ofmammalian leukocytes, but only expressed paramyosin (Ag-B),an exclusive cytoskeleton component of cestodes, nematodes andsome insects (Fig. 4A) [20–24]. This parasite cell population wasselected for further ER-like expression analyses. In contrast withspleen cells, which were CD3+/MHC class II+ (Fig. 4B), the ER-like protein was mainly recognized on T. crassiceps cells, whichwere CD3−/MHC class II− (Fig. 4C–E). Interestingly, the fluores-cence intensity related to the ER-like protein observed in E2-treatedparasites was 3-fold above the level observed in untreated controlcysticerci (Fig. 5).
3.5. ER-like protein is present in the sub-tegumental cells of T.crassiceps cysticerci
As mentioned above, experimental evidence indicated that ER-like protein is expressed in T. crassiceps cysticerci, and is not acontamination product from host cells. Thus, immunofluorescentassays on well-preserved T. crassiceps tissue were performed, andresults showed that parasite cells express ER-like protein mainly inthe subtegumental cysticercus tissue (Fig. 6C and D). As expected, T.crassiceps tissue incubated only in the presence of secondary anti-body gave no positive signal (Fig. 6B), which demonstrated thatthe experimental conditions were optimal for detecting exclusivelyparasite cells presenting ER-like molecules and not false positivesignals.
3.6. Identification of the T. crassiceps ER-like protein by 2DEblotting
After bidimensional electrophoresis of total proteins from
E2-treated parasites, the spots were resolved in the CoomassieBlue-stained gels (Fig. 7A). Most spots displayed neutral and alka-line pI with a wide range of MW, and some were well-recognized bypolyclonal antibodies as shown in Fig. 7B. Since well-characterized
1154 E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149– 1159
F ipitatm cipitata
eMwctpcs(
3
ofisb1casfo
FC
ig. 2. (A) Western blot of T. crassiceps ER-like protein and (B) specific immunoprecouse uterus ER protein were purified from total protein extracts. (B) Immunopre
nd non-treated cysticerci. Arrow indicates the ER protein.
strogen receptors have been localized around pH values of 6.0 andW values of 66 kDa, the possibility of this molecule being an ERas high. Based on antibody recognition, other ER-like isoforms
ould also perhaps be present in the same MW band of the iden-ified protein. Sequencing of the spot yielded a small fragment ofrotein similar to a mammalian nuclear estrogen receptor, whichorresponds to the translation product of the mRNA previouslyequenced by us (Gene Data Bank accession number AY596184)Fig. 7C).
.7. Phylogenetic analyses and Sequence alignment
A posterior analysis of the T. crassiceps protein showed homol-gy of around 60% to the protein sequences previously reportedor mouse, rat, rabbit and human ER-� in the Gene Data Bank. Its important to mention that the analyzed conserved motif wasituated in a region of approximately 90 aa, located in the DNA-inding domain of the C-terminal motif (from aa position number10 to 160 of the mammal sequences described above). A more pre-ise analysis of the T. crassiceps putative ER-� sequence involved
Neighbor Joint Tree (NJT) for studying phylogenetic relation-hips (Fig. 8). The partial sequence of the estrogen-binding proteinrom T. crassiceps obtained in the present study was aligned tother 13 sequences, conforming a data set of 396 aa. This NJT was
ig. 3. Comparison between complexity and size of T. crassiceps cells and mouse spleen characteristic mouse spleen showing normal size and complexity. SSC = Side scatter; FSC
ion of T. crassiceps ER-like protein. (A) ER-like protein from cultured cysticerci, andion assays showed that the ER-like protein expression level was similar in treated
inferred from the ER-� dataset, producing a single tree composedby 5 groups. The maximum parsimony tree inferred with this dataset, yielded single tree with a length = 701, and with a consistenceindex = 0.95. This unrooted tree (Fig. 8) was composed by 5 groups.The first contains sequences of 3 mammals (rat, human and naturalhost of the parasite) with strong bootstrap support of 100%. The sec-ond group consisted of one reptilian and one bird. The third groupincluded two sequences of amphibians. The fourth group includessequences of 3 species of fish. Finally the fifth group contains 3sequences of Platyhelminthes, including T. crassiceps. The phylo-genetic relationships among the 5 groups received good bootstrapsupport ranging from 66% to 100% (see Fig. 8). Additionally, T. cras-siceps ER-� is related to the ER-� family of vertebrates, more closelyassociated to reptilian and amphibian (Fig. 8). This finding suggeststhat T. crassiceps ER-� is definitively not a product of host cell con-tamination, specifically not of mouse or human cells, because of thebig distance between T. crassiceps and mammals in the NJT.
4. Discussion
As we reported previously, 17-�-estradiol exerts a direct pro-liferative effect on T. crassiceps cysticercus reproduction, which isnot necessarily mediated by the host’s immune system but by aclassic nuclear receptor in the parasite [12]. The aim of the present
ells. (A) Parasite cells show smaller size and similar complexity as spleen cells. (B) = Forward scatter. Results are representative of 3 experiments.
E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149– 1159 1155
Fig. 4. Specific detection of an ER-like protein in T. crassiceps. FACS analysis showed that the ER-like protein detected in T. crassiceps was not a contamination productfrom host immune CD3 and MCH class II positive cells. (A) Parasite cell paramyosin+, CD4−/CD8− . (B) Mouse spleen cells CD3+/MHC II+. (C) Basal inmmunofluorescence incysticerci cells with no added antibodies. (D) Untreated parasite ER-like protein+, CD3−/MHC II− . (E) Parasite cells treated with estradiol (40 �g/ml) ER-like+, CD3−/MHC II− .Controls = Parasites treated with the vehicle in which hormone and tamoxifen were dissolved. Results are representative of 3 experiments.
Fig. 5. ER-like protein expression in T. crassiceps cells. (A) Mean fluorescence intensity of ER-like protein expression in parasite cells. In non-treated cysticerci very few cellspresented a low immnofluorescent signal (thin line) while estradiol-treated cysticerci showed few cells with a high immnoflurescent signal related to the ER-like proteinexpression level (thick line). (B) Graphic representation of mean fluorescence intensity. Estradiol-stimulated cysticerci showed a 3-fold increase in fluorescence intensitycompared with control non-treated parasites. Results are representative of 3 experiments.
1156 E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149– 1159
Fig. 6. Localization of an ER-like protein in T. crassiceps by immunofluorescence. (A) Transversal section of one T. crassiceps cysticercus where tegument, sub-tegument andcells are observed with Nomarski. (B) Negative control of immunofluorescence using the secondary antibody. (C) Specific detection of ER-like protein (arrows) in parasitecells mainly located along subtegumental tissue, 10×. (D) A magnification of 40× from “C” (area boxed) shows details of the T. crassiceps cells expressing ER-like proteinexclusively on subtegumental cells (arrows). T, tegumental cells; GL, germinal layer.
Fig. 7. ER-like detection by 2D electrophoretic analysis. (A) Total protein of in vitro cultured T. crassiceps was resolved in a 2D gel. (B) A well-defined dot of 66 kDa, pI = 6.0was detected in T. crassiceps using an antibody to detect the protein (red circle). Molecular weight of proteins are as indicated in the figure. (C) Spot sequencing produced asmall fragment of protein similar to a mammalian nuclear estrogen receptor (Gene Data Bank accession number AY596184). Arrows indicate protein isoelectrofocusing (pH4–7) and their resolution in the second electrophoresis running using MW commercial standards as indicated. In A, the gel was stained by Coomassie Blue and in B, proteinswere resolved in chemiluminescence assays after their recognition by primary and secondary antibodies.
E.G. Ibarra-Coronado et al. / Steroids 76 (2011) 1149– 1159 1157
Fig. 8. Maximum parsimony tree inferred from a data set of 396 aa. Numbers near nodes show MP bootstrap frequencies. Neighbor Join Tree (NJT) for phylogenetic relationanalysis. ERs from several species of fish, amphibian, reptilian, bird and mammals were analyzed through a NJT for searching probable relationship to the T. crassiceps ERi fish
m
slrt
eimerimptBCstmdtwbeooIt
dentified and sequenced. The T. crassiceps protein showed close relation to ERs fromeans bootstrap support ranging among analyzed species.
tudy was to investigate the participation of an estrogen receptor-ike molecule in the helminth cestode T. crassiceps, with possibleeactiveness to endogenous estradiol which could be involved inhe parasite’s proliferative processes.
In earlier work, we found that gender and circulating E2 lev-ls in host mice crucially affect the dynamics of parasite loadsn mice infected with T. crassiceps cysticerci [25,26]. Infection of
ale mice with T. crassiceps leads to striking increments in hoststrogen levels, which is consistent with the notion that cysticercieproduce better in high estrogen conditions and somehow inducets production in the host. An inadequate hormonal environment
ay lead to apoptosis of crucial parasite cells, as has been pro-osed for other parasite infections, i.e., retinoic acid has been showno affect female Litomosoides carinii and microfilariae of L. carinii,rugia malayi, Brugia pahangi, and Acanthocheilonema viteae [27].ercariae, schistosomula, and adult worms of Schistosoma mansonihow reduced viability and significant inhibition of in vitro schis-osoma oviposition [28] while testosterone does likewise with the
itochondrial function of S. mansoni [29]. Because there is a greateal of conserved sequence homology among most hormone recep-ors, especially in the ligand and DNA-binding domains [30], weere able to show that cysticerci expresses an ER. Such similarities
etween host and cysticercus metabolism are not surprising sincextensive homologies between species are being documented in
ther systems as well. Interestingly, present results show that onlyne isoform of the classic ER (ER-�) is expressed in T. crassiceps.t appears that the binding of E2 to its respective receptor causeshe effects of estrogens. Binding of the ER to the classic estrogen-and amphibian, but distant to their counterparts in mammals. Numbers on the NJT
dependent elements could be responsible for the activation of AP-1complex genes in the normal metabolism of T. crassiceps. Previousstudies have demonstrated that the genome of O. volvulus encodesat least 3 members of the nuclear receptor family [31], and thiscould also be the case for T. crassiceps.
The present study revealed that progressively higher 17-�-estradiol concentrations correlate with the number of T. crassicepscysticercus buds. The opposite response was observed when acompetitive inhibitor of estradiol-binding to the ER (tamoxifen)was tested in culture. As tamoxifen concentration increased, par-asite reproduction progressively decreased. On the other hand,the effect of E2 was enhanced as time passed, reaching its maxi-mum effect on parasite reproduction at day five of in vitro culture,which supports the fact that E2 effects depended on its con-centration. Nevertheless, no time-dependent response was foundwhen tamoxifen was tested, although complete blockage of theE2 dependent proliferative effect occurred since the first day sug-gesting that the parasite ER-like protein is directly involved inmediating the cross-talk between the hormonal microenviron-ment (exogenous E2) and parasite reproduction. These findingssupport previous results showing a marked concentration andtime-dependent pattern regarding the effects of E2 on cysticercusreproduction. These observations are relevant since they suggestthat sex-steroids may have similar effects on mammals and on
parasitic cestodes, a hypothesis that evokes the wide range ofeffects of steroid hormones not only on several different cell types,but also along the phylogenetic scale, among distantly relatedorganisms.
1 / Ster
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158 E.G. Ibarra-Coronado et al.
Moreover, determination of the effects of E2 and tamoxifen on T.rassiceps cysticerci allowed to determine the expression and trans-ation of the ER-like gene into functional proteins which mediatehe E2 effects. Based on this, a band corresponding to the ER-likeene was amplified from T. crassiceps larval tissue by means of spe-ific primers designed considering the most conserved regions ofhis gene, which have been previously reported in mammals such ashe mouse, rat and human, as well as in yeast, birds and amphibians.urthermore, not only is the ER-like gene expressed in the par-site, but it can also be significantly downregulated by tamoxifen.his unexpected finding suggests that helminth parasites may haveeveloped a positive feed-back mechanism able to sense changes
n the expression of very important molecules (such as ER) andaintain their activity in order to avoid compromising the viabil-
ty and reproduction of the parasite. Also, this finding offers anlternative explanation to the finding that T. crassiceps cysticercirow better in female mice than in male mice [12], and emphasizeshe molecular cross-talk between host and parasite, which, in turn,s differentially influenced by the hormonal microenvironment ofach gender.
A critical aspect of the present study was to determine thathe detected and analyzed ER-like protein was exclusively foundn the T. crassiceps cysticercus, and it was not a consequence ofost immune cell contamination, because, as shown elsewhere,here is extremely high interaction between parasites and immuneells, which may eventually lead to leukocyte invasion of severalarasitic tissues [32]. For this reason, an alternative use of flowytometry analysis was used to differentiate proteins from T. crassi-eps and the murine host by identifying molecules exclusive of thearasite that are neither synthesized nor expressed by the host.his is the case of paramyosin, a muscle protein found only innvertebrates such as Drosophila melanogaster, Caenorhabditis ele-ans, T. solium and T. saginata [33]. The flow cytometry studieshowed the presence of a specific ER-like protein in the parasite,ecause paramyosin was only detected in T. crassiceps cells, wherehis estrogen receptor-like molecule was also found and studied.n contrast, the anti-paramyosin antibody did not recognize cellsxtracted from mouse spleen, which in addition were positive forD4 and MHC class II antibodies, contrary to parasite cells.
These results show that the origin of the analyzed ER-like pro-ein was in fact T. crassiceps, and simultaneously demonstrate theotential use of flow cytometry for differential identification ofolecules from organisms which are in extremely close contact,
uch as host and parasite.The fact that the parasite ER-like protein differs from its homo-
ogue in mammalian cells supports two very important aspectsf this study: (a) the parasite ER-like molecule is not a productrom host immune or epithelial cells, and (b) although the two pro-eins differ in some characteristics, they probably conserve a highegree of similarity in their catalytic domains and are thus proba-ly detectable with the same antibody. It is important to mentionhat the ER-like protein was not only detected at the mRNA androtein levels, but it was also localized inside the parasite cell. Inter-stingly, parasite cells expressing ER-like protein were exclusivelyocated in the subtegumental tissue, where most of the muscle andephridium cells are found. This suggests that the ER-like protein islso involved in parasite motility and excretion, as well as in repro-uctive functions, the two of which together are responsible foraintaining parasite viability and proliferation.Our hypothesis supports the fact that the expression of parasite
roteins which can recognize the host’s growth factors representsn advantage in parasite metabolism economy, since the pathogen
oes not need to synthesize all molecules involved in a pathway,ut can use them directly from its host. This benefits the processesf reproduction, establishment and immune evasion, among othermportant aspects of the parasite’s life.[
oids 76 (2011) 1149– 1159
Regarding the significance of the differential effects of sex hor-mones on host and parasite, it is interesting to speculate on theevolutionary impact of host gender distinctive specialization indealing with the parasite’s developmental stages. Thus, it wouldappear that gender specialization as the one described here forT. crassiceps (females favoring and males hindering asexual larvalreproduction) has contributed to the evolution of a more stablehost–parasite relationship.
Furthermore, this protein in T. crassiceps showed high degreeof relation to their ER-� counterparts in fish and amphibian, butit is distant to mammalian sequences. This finding has two impor-tant connotations: first, it suggests that ER from T. crassiceps is aclose relative of the steroid nuclear receptors that bind to estrogens.Second, this ER in T. crassiceps definitively is not a contaminationproduct from mouse or human cells because it has a far relation toERs sequenced in these organisms.
In conclusion, a functional ER-like protein from T. crassiceps ispresently described. This ER-like molecule showed great capac-ity to transduce signals induced by 17-�-estradiol in the parasite.These results provide further evidence on the mechanism of howthe host’ microenvironment affects parasite metabolism. Further-more, the evolutionary origin of the molecules described herein,which involves the use of the host’s hormones by the parasite, isworth studying. Whether the genes that code for these moleculeswere acquired by the parasite through horizontal gene transferor evolved by mimicry, or simply from common ancestral genes,remains to be elucidated. Finally, our findings provide evidence onthe cross-talk between host and parasite at molecular and evolu-tionary levels, and they corroborate the sexual dimorphism of theimmune response, providing new information which may be use-ful in designing anti-helminth drugs able to combat parasite cellsspecifically with minimal secondary effects to the host.
Acknowledgements
Financial support was provided by Grant # IN213108 fromPrograma de Apoyo a Proyectos de Innovación Tecnológica, Direc-ción General de Asuntos del Personal Académico, (PAPIIT, DGAPA),UNAM to J. Morales-Montor. Romel Hernández-Bello holds apostdoctoral fellowship from DGAPA, UNAM., and Elizabeth G.Ibarra-Coronado holds a scholarship from CONACYT, México. IsabelPérez Montfort corrected the English version of this manuscript.
References
[1] Olster DH, Blaustein JD. Development of steroid-induced lordosis in femaleguinea pigs: effects of different estradiol and progesterone treatments, cloni-dine, and early weaning. Horm Behav 1989;23:118–29.
[2] Pfaff DW, Freidin MM, Wu-Peng XS, Yin J, Zhu YS. Competition for DNA steroidresponse elements as a possible mechanism for neuroendocrine integration. JSteroid Biochem Mol Biol 1994;49:373–9.
[3] Camacho-Arroyo I, Guerra-Araiza C, Dominguez R, Mendoza-Rodriguez CA,Cruz ME, Cerbon MA. C-fos expression pattern in the hypothalamus and thepreoptic area of the rat during proestrus. Life Sci 1998;62:1153–9.
[4] Guerra-Araiza C, Cerbon MA, Morimoto S, Camacho-Arroyo I. Progesteronereceptor isoforms expression pattern in the rat brain during the estrous cycle.Life Sci 2000;66:1743–52.
[5] Choi KC, Jeung EB. The biomarker and endocrine disruptors in mammals. JReprod Dev 2003;49:337–45.
[6] An SJ, Zhang YX. Estrogen receptor subtypes and the regulatory effect ofreceptor ligand binding on gene transcription. Sheng Li Ke Xue Jin Zhan2002;33:309–12.
[7] Okada A, Sato T, Ohta Y, Iguchi T. Sex steroid hormone receptors in thedeveloping female reproductive tract of laboratory rodents. J Toxicol Sci2005;30:75–89.
[8] Charitidi K, Meltser I, Tahera Y, Canlon B. Functional responses of estrogen
receptors in the male and female auditory system. Hear Res 2009;252:71–8.[9] Greene GL, Shiau AK, Nettles KW. A structural explanation for ERalpha/ERbetaSERM discrimination. Ernst Schering Res Found Workshop 2004:33–45.
10] Henke BR, Heyer D. Recent advances in estrogen receptor modulators. CurrOpin Drug Discov Dev 2005;8:437–48.
/ Ster
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
cellular responses. Exp Parasitol 2002;100:235–47.[33] Maroto M, Arredondo JJ, San Roman M, Marco R, Cervera M. Analysis of the
E.G. Ibarra-Coronado et al.
11] Morales-Montor J, Chavarria A, De Leon MA, Del Castillo LI, Escobedo EG,Sanchez EN, et al. Host gender in parasitic infections of mammals: an evaluationof the female host supremacy paradigm. J Parasitol 2004;90:531–46.
12] Escobedo G, Larralde C, Chavarria A, Cerbon MA, Morales-Montor J. Molecularmechanisms involved in the differential effects of sex steroids on the repro-duction and infectivity of Taenia crassiceps. J Parasitol 2004;90:1235–44.
13] Vargas-Villavicencio JA, Larralde C, Morales-Montor J. Treatment with dehy-droepiandrosterone in vivo and in vitro inhibits reproduction, growth andviability of Taenia crassiceps metacestodes. Int J Parasitol 2008;38:775–81.
14] Escobedo G, Camacho-Arroyo I, Hernandez-Hernandez OT, Ostoa-Saloma P,Garcia-Varela M, Morales-Montor J. Progesterone induces scolex evagina-tion of the human parasite Taenia solium: evolutionary implications to thehost–parasite relationship. J Biomed Biotechnol 2010;2010:591079.
15] Escobedo G, Soldevila G, Ortega-Pierres G, Chavez-Rios JR, Nava K, Fonseca-Linan R, et al. A new MAP kinase protein involved in estradiol-stimulatedreproduction of the helminth parasite Taenia crassiceps. J Biomed Biotechnol2010;2010:747121.
16] Vargas-Villavicencio JA, Larralde C, De Leon-Nava MA, Escobedo G, Morales-Montor J. Tamoxifen treatment induces protection in murine cysticercosis. JParasitol 2007;93:1512–7.
17] Townson S, Tagboto SK. In vitro cultivation and development of Onchocercavolvulus and Onchocerca lienalis microfilariae. Am J Trop Med Hyg1996;54:32–7.
18] Morales-Montor J, Escobedo G, Rodriguez-Dorantes M, Tellez-Ascencio N, Cer-bon MA, Larralde C. Differential expression of AP-1 transcription factor genesc-fos and c-jun in the helminth parasites Taenia crassiceps and Taenia solium.Parasitology 2004;129:233–43.
19] Hernandez-Bello R, Bermudez-Cruz RM, Fonseca-Linan R, Garcia-Reyna P, LeGuerhier F, Boireau P, et al. Identification, molecular characterisation and dif-ferential expression of caveolin-1 in Trichinella spiralis maturing oocytes andembryos. Int J Parasitol 2008;38:191–202.
20] Vargas-Parada L, Laclette JP. Gene structure of Taenia solium paramyosin. Par-asitol Res 2003;89:375–8.
21] Yang J, Yang Y, Gu Y, Li Q, Wei J, Wang S, et al. Identification and characteriza-tion of a full-length cDNA encoding paramyosin of Trichinella spiralis. BiochemBiophys Res Commun 2008;365:528–33.
22] Erttmann KD, Buttner DW. Immunohistological studies on Onchocerca volvulusparamyosin. Parasitol Res 2009;105:1371–4.
oids 76 (2011) 1149– 1159 1159
23] Jiz M, Friedman JF, Leenstra T, Jarilla B, Pablo A, Langdon G, et al. Immunoglob-ulin E (IgE) responses to paramyosin predict resistance to reinfectionwith Schistosoma japonicum and are attenuated by IgG4. Infect Immun2009;77:2051–8.
24] Liu H, Miller MS, Swank DM, Kronert WA, Maughan DW, Bernstein SI.Paramyosin phosphorylation site disruption affects indirect flight muscle stiff-ness and power generation in Drosophila melanogaster. Proc Natl Acad Sci USA2005;102:10522–7.
25] Larralde C, Morales J, Terrazas I, Govezensky T, Romano MC. Sex hormonechanges induced by the parasite lead to feminization of the male host inmurine Taenia crassiceps cysticercosis. J Steroid Biochem Mol Biol 1995;52:575–80.
26] Morales-Montor J, Baig S, Hallal-Calleros C, Damian RT. Taenia crassiceps: andro-gen reconstitution of the host leads to protection during cysticercosis. ExpParasitol 2002;100:209–16.
27] Zahner H, Sani BP, Shealy YF, Nitschmann A. Antifilarial activities of syntheticand natural retinoids in vitro. Trop Med Parasitol 1989;40:322–6.
28] Morales-Montor J, Mohamed F, Ghaleb AM, Baig S, Hallal-Calleros C, DamianRT. In vitro effects of hypothalamic–pituitary–adrenal axis (HPA) hormones onSchistosoma mansoni. J Parasitol 2001;87:1132–9.
29] Fantappie MR, Galina A, Luis de Mendonca R, Furtado DR, Secor WE, Colley DG,et al. Molecular characterisation of a NADH ubiquinone oxidoreductase subunit5 from Schistosoma mansoni and inhibition of mitochondrial respiratory chainfunction by testosterone. Mol Cell Biochem 1999;202:149–58.
30] Damian RT. Parasite immune evasion and exploitation: reflections and projec-tions. Parasitology 1997;115(Suppl):S169–75.
31] Unnasch TR, Bradley J, Beauchamp J, Tuan R, Kennedy MW. Characterization ofa putative nuclear receptor from Onchocerca volvulus. Mol Biochem Parasitol1999;104:259–69.
32] Rajan TV, Ganley L, Paciorkowski N, Spencer L, Klei TR, Shultz LD. Brugian infec-tions in the peritoneal cavities of laboratory mice: kinetics of infection and
paramyosin/miniparamyosin gene. Miniparamyosin is an independently tran-scribed, distinct paramyosin isoform, widely distributed in invertebrates. J BiolChem 1995;270:4375–82.
