Research Article The Involvement of the Androgen Receptor...

7
Research Article The Involvement of the Androgen Receptor in the Secretion of the Epididymal corpus in the Lizard Podarcis sicula Mariailaria Verderame Department of Biology, University of Naples Federico ΙΙ, Via Mezzocannone 8, 80134 Naples, Italy Correspondence should be addressed to Mariailaria Verderame; [email protected] Received 25 September 2013; Accepted 9 December 2013; Published 29 January 2014 Academic Editor: Roger P. Croll Copyright © 2014 Mariailaria Verderame. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A crucial role in the maintenance of male reproductive functions is carried out by the androgen through its receptor in balance with the estrogen receptors (ERs). e distribution of the androgen receptor (AR) is well documented in the testis and in the reproductive tissues of mammals but the findings about the AR in nonmammalian vertebrates and in particular in reptiles are very scarce. Here by means of in situ hybridization (ISH) we investigated the AR expression along the epididymal channel (efferent ductules, corpus, and cauda) of Podarcis sicula during the mating and nonmating period. e results show that in this seasonal breeding species the AR expression pattern is always constant throughout the epididymis. e administration of estradiol-17 in the mating period does not affect the AR expression but inhibits the secretory activity of the epididymal corpus. To verify the expression pattern of ERs, we also conducted ISH investigations on adjacent sections with ERs probes. e findings suggest that AR induces the secretory activity in the epithelial cells of the epididymal corpus and confirm our previous results that showed the role of ERalpha (ER) as switch off for the secretion of this compartment. 1. Introduction e epididymis can be regionalized, depending on the species, into the initial segment or caput which includes the efferent ductules, the middle piece or corpus characterized by a high secretory activity, and terminal segment or cauda. In these compartments the maturation of spermatozoa takes place before ejaculation [14]. e majority of studies about the regulation of epididymal functions concern the expression of the steroid hormone receptors, that is, androgen (AR) and/or estrogen (ER and ER) receptors in mammals [510]. In nonmammalian vertebrates, little is known about the expression of AR in the epididymis [11, 12]. In this paper the expression of the androgen receptor is detected in the lizard Podarcis sicula. e histological structure of the Podarcis epididymis, the evolution of its reproductive cycle, and the pattern of steroid hormones have been already described in detail [1315]. Briefly, in this lizard the epididymis is a channel of 7-8mm in length and has been divided into three regions according to the morphological and functional characteristics of the surrounding epithelium [15]. In the region defined caput are including small efferent ductules with ciliate cubic epithelium that does not change during the year [15]. Conversely, notable changes are found in the epithelium of the middle piece defined corpus. is epithelium is constituted by cubic cells during the winter reproductive stasis when the lumen is empty. During the mating period the epithelium becomes cylindrical and the cells appear elongated, oſten, binucleated, and engaged in a massive secretory activity. In this period the lumen is filled with spermatozoa and secretory granules that pass in the third region, cauda, whose epithelium does not show any changes throughout the year. e lumen of the cauda is empty during the nonreproductive season [15]. In Podarcis, we recently found that the expression of ER may act as a switch off for the secretory activity of the epididymal corpus [15]. Now by means of in situ hybridization (ISH) with the homologous Podarcis AR probe just obtained, it is demon- strated that in this lizard the AR expression is continuously present along the epididymis also in animals experimentally Hindawi Publishing Corporation International Journal of Zoology Volume 2014, Article ID 457830, 6 pages http://dx.doi.org/10.1155/2014/457830

Transcript of Research Article The Involvement of the Androgen Receptor...

Page 1: Research Article The Involvement of the Androgen Receptor ...downloads.hindawi.com/journals/ijz/2014/457830.pdfThe Involvement of the Androgen Receptor in the Secretion of the Epididymal

Research ArticleThe Involvement of the Androgen Receptor in the Secretion ofthe Epididymal corpus in the Lizard Podarcis sicula

Mariailaria Verderame

Department of Biology University of Naples Federico ΙΙ Via Mezzocannone 8 80134 Naples Italy

Correspondence should be addressed to Mariailaria Verderame ilariaverderameuninait

Received 25 September 2013 Accepted 9 December 2013 Published 29 January 2014

Academic Editor Roger P Croll

Copyright copy 2014 Mariailaria Verderame This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

A crucial role in themaintenance of male reproductive functions is carried out by the androgen through its receptor in balance withthe estrogen receptors (ERs)Thedistribution of the androgen receptor (AR) is well documented in the testis and in the reproductivetissues of mammals but the findings about the AR in nonmammalian vertebrates and in particular in reptiles are very scarce Hereby means of in situ hybridization (ISH) we investigated the AR expression along the epididymal channel (efferent ductules corpusand cauda) of Podarcis sicula during the mating and nonmating period The results show that in this seasonal breeding species theAR expression pattern is always constant throughout the epididymisThe administration of estradiol-17120573 in themating period doesnot affect the AR expression but inhibits the secretory activity of the epididymal corpus To verify the expression pattern of ERs wealso conducted ISH investigations on adjacent sections with ERs probesThe findings suggest that AR induces the secretory activityin the epithelial cells of the epididymal corpus and confirm our previous results that showed the role of ERalpha (ER120572) as switchoff for the secretion of this compartment

1 Introduction

The epididymis can be regionalized depending on thespecies into the initial segment or caput which includes theefferent ductules the middle piece or corpus characterizedby a high secretory activity and terminal segment or caudaIn these compartments the maturation of spermatozoa takesplace before ejaculation [1ndash4]

Themajority of studies about the regulation of epididymalfunctions concern the expression of the steroid hormonereceptors that is androgen (AR) andor estrogen (ER120572 andER120573) receptors in mammals [5ndash10]

In nonmammalian vertebrates little is known about theexpression of AR in the epididymis [11 12]

In this paper the expression of the androgen receptor isdetected in the lizard Podarcis sicula

The histological structure of the Podarcis epididymis theevolution of its reproductive cycle and the pattern of steroidhormones have been already described in detail [13ndash15]Briefly in this lizard the epididymis is a channel of 7-8mmin length and has been divided into three regions according

to the morphological and functional characteristics of thesurrounding epithelium [15] In the region defined caput areincluding small efferent ductules with ciliate cubic epitheliumthat does not change during the year [15] Conversely notablechanges are found in the epithelium of the middle piecedefined corpus This epithelium is constituted by cubic cellsduring the winter reproductive stasis when the lumen isempty During the mating period the epithelium becomescylindrical and the cells appear elongated often binucleatedand engaged in a massive secretory activity In this periodthe lumen is filled with spermatozoa and secretory granulesthat pass in the third region cauda whose epithelium doesnot show any changes throughout the year The lumen of thecauda is empty during the nonreproductive season [15] InPodarcis we recently found that the expression of ER120572 mayact as a switch off for the secretory activity of the epididymalcorpus [15]

Now by means of in situ hybridization (ISH) with thehomologous Podarcis AR probe just obtained it is demon-strated that in this lizard the AR expression is continuouslypresent along the epididymis also in animals experimentally

Hindawi Publishing CorporationInternational Journal of ZoologyVolume 2014 Article ID 457830 6 pageshttpdxdoiorg1011552014457830

2 International Journal of Zoology

treated in the mating period with estradiol-17120573 (E2) This

expression may be related to the morphophysiology of theepididymal channel during the annual reproductive cycle Tocompare the AR expression to the known ERs fluctuation[15] ISH with ER and ER120573 probes on adjacent section wasperformed

2 Material and Methods

21 Animals Adult males of lizard Podarcis sicula (about75ndash8 cm snout vent) were captured near Naples (Italy) fortwo consecutive years during the mating period (spring-early summer) [119899 = 8] and winter stasis (fall-winter) [119899 =8] kept in terraria at natural temperature and photoperiodand fed ad libitum with larvae of mealworm Some samples(119899 = 6) from the mating period were experimentally treatedintraperitoneally with estradiol-17120573 (168 ng100 120583L) in reptilephysiological solution (NaCl 007) every other day for 2weeks [15] In parallel three males were intraperitoneallyinjected with physiological solution (100 120583L) every other dayfor 2 weeks The animals were killed by decapitation afteranaesthesia in ice and the testes with the attached epididymiswere immediately excised and processed for the plannedanalyses All efforts were made to avoid animal suffering

Authorization to capture the animals for experimentaltreatments was granted by the Italian Ministry of the Envi-ronment (Auth SCN2D20009213)

22 Digoxigenin-Labeled Probes cDNA for AR was obtainedby RT-PCR from the total Podarcis testis RNA and amplifiedwith forward primer 51015840-TGGGCAACCTGAAGATGC-31015840and reverse primer 51015840-ACCCCATGGCGAAAATCAT-31015840designed on the known AR sequences of some reptiles Tri-meresurus flavoviridis (AB5483001) Elaphe quadrivirgata(AB5483011) Anolis carolinensis (AF2232242) Calotesversicolor (AF2753702) Trachemys scripta (DQ8489891)Pseudemys nelsoni (AB3010611) Cnemidophorus uniparens(S799381) Alligator mississippiensis (AB1863562) andLeiolepis reeversii rubritaeniata (AB4903851)

First strand cDNA (3 120583L) was used as template in PCRamplification (final volume 25mL) The PCR thermal settingwas as follows 4 min at 94∘C 38 cycles of 30 s at 94∘C 40 sat 50∘C 1min at 72∘C 7min at 72∘CThe obtained fragmentwas sequenced using the BigDye Terminator v11 CycleSequencing Kit (Applied Biosystems) and run on the ABIPRISM 310 Genetic Analyzer and compared to GeneBankdatabase

The ER120572 and ER120573 probes were obtained as alreadydescribed [15 16]

23 Histology and In Situ Hybridization (ISH) The epi-didymis excised from the testes was fixed in Bouinrsquos fluid[17] dehydrated in alcohol increasing gradation clarified inxylene and embedded in paraffin Sections 7 120583m in thicknesswere obtained with Reichert-Jung 2030 microtome Somesections were stained with Malloryrsquos trichrome modified byGalgano [17]

The ISH was performed on adjacent sections with ARER120572 or ER120573 probes and was carried out as described in theliver and epididymis [15 18] Briefly the dewaxed sectionswere treated with proteinase K (10 120583gmL) at 50∘C for 10minDigoxigenin labelled probes were used at a concentrationof 80 ng100 120583L in hybridization buffer [Tris-HCl 002MpH 75 NaCl 03M EDTA 001M DTT 01M formamide50 Denhardtrsquos 1X tRNA 100 120583gmL ss-DNA 100 120583gmL]overnight at 50∘C in a moist chamber The slides wereincubated with RNasi mix at 37∘C for 30min and in thesame mix without RNasi at 37∘C for 30min washed in 2xSSC for 3min in 01x SSC at 60∘C 15min and in NTP andthen incubated in 2 blocking solution [Roche DiagnosticsMannheim Germany] in maleic acid buffer [01M maleicacid 015M NaCl pH 75] for 1 h The sections were keptovernight at 4∘C with an alkaline phosphatase-conjugatedsheep anti-DIG antibody (Roche Diagnostics) (1 2500) inblocking solution and rinsed in NTP [Tris-HCl 01M pH75 NaCl 015M] buffer for 30 min and in NTM buffer[Tris-HCl 100mM pH 95 MgCl 50mM NaCl 100mM] for30min Finally the sections were kept in the dark at roomtemperature in the colour detection substrate solution nitrob-lue tetrazolium and 5-bromo-4-chloro-31015840-Indolylphosphate(BCIPNBT) as recommended by manufacturerrsquo (Roche) inNTM until appearance of the colour To exclude a cross-linkwith genomicDNA some other adjacent sectionswere treatedwith DNAsi Control sections were obtained by omittingincubation with the probes

3 Results

31 Sequencing of Podarcis AR The product of reverse-trans-criptase-PCR from Podarcis testis gave a single band of about300 bp in size (Figure 1(a)) BLAST analysis of the obtainednucleotide sequence (NCBI data bank) revealed high identity(83minus79) to the AR sequences reported in several reptiles(Figures 1(b) and 1(c)) This partial sequence of PodarcisAR cDNA was annotated in EMBL Gen Bank (Accessionnumber JQ219668)

32 ISH with AR Probe The homologous AR probe gavepositive signals continuously throughout the mating periodandwinter stasis in the efferent ductules in the corpus (Figures2(a) and 2(c)) and in the cauda (one for all Figure 2(b))

The epididymal tract of samples treated with estradiol 17-120573 (E2) in the full mating period showed the expression of AR

in the efferent ductules (data not shown) corpus (Figure 1(d))and cauda (data not shown) as in the natural samples Controlanimals treated with physiological solution showed the sameexpression already described for the untreated males (datanot shown)

The sections incubated by omitting the AR probe gavealways negative results (data not shown)

33 ISH with ERs Probe The ISH results on ERs expressionconfirm what has already been found by us in the epididymisof Podarcis [15] In the mating period the ER120572 expressiondid not occur in the secreting corpus (Figure 3(a)) but was

International Journal of Zoology 3

300bp

(a)

Androgen receptors in reptiles1 300

Podarcis156 457Anolis

83

82

82

81

83

83

82

82

82

79

165 466Leiolepis

Calotes 167 468

Cnemid uni 46 348

Trimeresurus 294 594

Protobothrops 153 455

Elaphe 294 594

Trachemys 49 349

Pseudemys 1517 1816

Pelodiscus 94 394

(b)

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

223

234

360

69

115

230

114

283

294

420

129

175

290

174

343

354

480

189

235

350

234

403

414

540

249

295

410

294

463

474

600

300

355

470

354

Podarcis versus other reptiles163

174

300

10

55

170

54

(c)

Figure 1 Sequencing of androgen receptor (a) Agarose gek of RT-PCR on cDNA of Podarcis testis (b) Similarity of predicted amino acidsequences of many reptiles with Podarcis AR (c) BLAST alignment of the nucleotide sequence of AR in Podarcis compared to other reptilesas Anolis Calotes Trimeresurus Trachemys Leiolepis and Cnemidophorus

evident in the efferent ductules (Figure 3(a)) and cauda (datanot shown) During the winter stasis the ER120572 expressionremained evident in the efferent ductules (Figure 3(b)) andcauda (data not shown) and occurred in the corpus devoidof secretion in this period (Figure 3(b))

The ER120573 probe gave positive signals continuouslythroughout the mating period and winter stasis in all theepididymal compartments (data not shown)

In the animals treated in the mating period with E2

an intense ER120572 expression took place in the corpus andthe secretory activity resulted inhibited the efferent ductulesand cauda were still positive (data not shown) ER120573-mRNAdespite the E

2treatment was unchanged compared with

untreated animals (Figure 3(c))

4 Discussion

The epididymis of the lizard Podarcis sicula is characterizedby a cyclic secretion that occurs in the corpus during themating period when this compartment is characterized by acylindrical epithelium producing a great amount of secretorygranules released into the enlarged lumen where many sper-matozoa are travelling from the rete testis In the winter stasisthe cuboidal epithelium surrounds a small totally emptylumen [15] Our previous results demonstrated that this

cyclic secretory activity may be negatively regulated by theexpression of ER120572 In the lizards treated with E

2during the

mating period the epididymal structure becomes equivalentto that observed during the winter stasis the epithelium isreduced in height and the secretory activity stops [15]

The present findings reveal that in Podarcis the epi-didymis may be also androgen dependent and the expressionof AR is constant during the reproductive cycle and along thewhole epididymal length efferent ductules corpus and cauda

The dependence of the epididymis on androgens isknown [8] and in several mammalian species the presence ofAR has been widely demonstrated by variousmethodologicalapproaches [6 19ndash25] In mouse and rat the AR is expressedin a cell-type-specific manner and the protein expression ishigher in the caput and corpus [9] AR-mRNAand protein arealso present in the human with a falling concentration fromthe caput to the cauda [8] It is also known that the androgenscan autoregulate the expression of AR-mRNA promoting ARprotein stabilization too [26]

Among nonmammalian vertebrates the AR protein wasfound in the epithelial cells lining the efferent ductules and inthe epididymal tubules of cockerel [27]

In rooster and drakes a different sensitivity of the epididy-mal segments to androgen has been suggested [11] In reptilesimmunoreactive AR was recorded only in the epididymis ofgreen turtle Chelonia mydas [12]

4 International Journal of Zoology

(a) (b) (c)

(d) (e)

Figure 2 Changes in themorphology andunchanged expression ofAR in the epididymal channel ofPodarcis in the natural cycle or in samplestreated with E

2during the mating period (a) Mating period positive signal for AR-mRNA in the epithelium of the efferent ductules (uarr) and

corpus (lowast) (b) Staining with Malloryrsquos modified trichrome-mating period note the ciliated cubic cells surrounding the efferent ductules (uarr)and the elongated actively secreting cells of the corpusin which lumen is filled with secretory granules (red) and spermatozoa (dark blu) (c)Mating period positivity for AR-mRNA in the cauda (d) Winter stasis both the efferent ductules (uarr) and the corpus (lowast) are still positive (e)The nonsecretory epithelium of the corpus (lowast) expresses AR-mRNA also in E

2treated males in the mating period (lowast) The bar is 30 120583m

In Podarcis it is now shown that the AR-mRNA iscontinuously expressed in the efferent ductules corpus andcauda regardless of the reproductive moment of this seasonalbreederThese results seem in contrast with the plasma levelsof testosterone that reach the maximum during the matingperiod and falls down at the end [14] Since the endotesticularprofile of testosterone shows significant level at the end ofmating remaining discrete throughout the reproductive cycle[14] it is possible that the constant presence of AR-mRNAmay be related to the intratesticular level of androgen even ifat different concentration

The simultaneous presence of several receptors in thesame cells raises questions about the receptors cross talkin the male reproductive system The coexistence of theandrogen and estrogen receptors (ER120572 and ER120573) in malegonad has been discussed in mammals [22 24 28 29]and among the nonmammalian vertebrates only in theturtle [12] In mouse the distribution of ER120573 in the epi-didymis was similar to that of AR [28] In rat ER120573 isconstitutively expressed in the efferent ductules while ARand ER120572 are selectively modulated by their own ligand [24]Physical interactions between AR and ER120572 resulting in

the estradiol-induced modulation of AR transcriptionalactivity have been described to indicate receptors interplayThese interactions arise between the C-terminal ER120572 ligand-binding domain and the N-terminal AR transactivationaldomain or with the full-length AR ER120573 did not interactwith AR [30] Furthermore multiple consensus sequences inthe hamster AR promoter region recognizes as transcriptionfactors ER or AR itself [31]

We recently described in Podarcis that the expression ofER120572 stops during the matings when the cells of the corpus areinvolved in its secretory activity and occurs after E

2treatment

that inhibits the secretion ER120573 expression does not changeneither after E

2treatment [15] The present observations

confirming these pathways for the ERs show at same timethe constant expression of AR throughout the reproductivecycle even in the animals treated with E

2 On the basis

of these results it is possible to suggest that in PodarcisAR expression induces the secretion in the cells lining thecorpus when the ER120572 expression does not occur Converselyduring the winter stasis and in E

2treated males when the

secretory activity of the corpus is inhibited AR and ER120572 arecoexpressed

International Journal of Zoology 5

(a) (b) (c)

Figure 3 ISH with Er120572 or ER120573 homologous probes on the epididymis (a) Mating period Er120572-mRNA is evident in the epithelium of theefferent ductules (uarr) no expression in the secretory epithelium of the corpus (lowast) Note in the corpus the enlarged lumen (lowast) (b) Winter stasisthe Er120572 expression is evident in the efferent ductules (uarr) and in nonsecreting corpus (lowast) (c) E

2treated males in mating period ER120573-mRNA

is expressed in the corpus (lowast) and in the efferent ductules (uarr) Note the restricted empty lumen of the corpus (lowast) The bar is 30 120583m

5 Conclusions

In conclusion in Podarcis AR is expressed in a constantmanner in the whole epididymal length throughout thereproductive annul cycle while the expression of ER120572 iscyclic in the corpus In particular it is possible to arguethat the secretory activity of the epididymal corpus can bepromoted by AR and inhibited by ER120572 The role of ER120573whose expression does not change neither after E

2treatment

remains Unsolved ER120573 may be constitutively expressed andcould ensure the activity of AR by modulating the expressionof ER120572

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The author is grateful to her tutor and teacher ProfessorErmelinda Limatola for critical revision of the paper

References

[1] M L Meistrich T J Hughes and W R Bruce ldquoAlterationof epididymal sperm transport and maturation in mice byestrogen and testosteronerdquo Nature vol 257 no 5531 pp 145ndash147 1975

[2] H DMoore and J M Bedford ldquoShort-term effects of androgenwithdrawal on the structure of different epithelial cells in therat epididymisrdquo Anatomical Record vol 193 no 2 pp 293ndash3111979

[3] S Carreau ldquoEstrogens-male hormonesrdquo Folia Histochemica etCytobiologica vol 41 no 3 pp 107ndash111 2003

[4] B Robaire S Seenundun M Hamzeh and S Lamour ldquoAndro-genic regulation of novel genes in the epididymisrdquoAsian Journalof Andrology vol 9 no 4 pp 545ndash553 2007

[5] R A Hess D Bunick K-H Lee et al ldquoA role for oestrogensin the male reproductive systemrdquoNature vol 390 no 6659 pp509ndash512 1997

[6] G Pelletier ldquoLocalization of androgen and estrogen receptorsin rat and primate tissuesrdquo Histology and Histopathology vol15 no 4 pp 1261ndash1270 2000

[7] C A Oliveira K Carnes L R Franca and R A Hess ldquoInfer-tility and testicular atrophy in the antiestrogen-treated adultmale ratrdquo Biology of Reproduction vol 65 no 3 pp 913ndash9202001

[8] N Ezer and B Robaire ldquoAndrogenic regulation of the structureand functions of the epididymisrdquo in The Epididymis FromMolecules to ClInical Practice B Robaire and B Hinton Edspp 297ndash316 Kluwer AcademicPlenum Publisher New YorkNY USA 2002

[9] S Yamashita ldquoLocalization of estrogen and androgen receptorsin male reproductive tissues of mice and ratsrdquo AnatomicalRecord A vol 279 no 2 pp 768ndash778 2004

[10] B Robaire and M Hamzeh ldquoAndrogen action in the epi-didymisrdquo Journal of Andrology vol 32 no 6 pp 592ndash599 2011

[11] R A P Dornas A G Oliveira M O Dias G A B Mahechaand C A Oliveira ldquoComparative expression of androgenreceptor in the testis and epididymal region of roosters (Gallusdomesticus) and drakes (Anas platyrhynchos)rdquo General andComparative Endocrinology vol 155 no 3 pp 773ndash779 2008

[12] S Otsuka M Suzuki N Kamezaki et al ldquoGrowth-relatedchanges in histology and immunolocalization of steroid hor-mone receptors in gonads of the immature male green turtle(Chelonia mydas)rdquo Journal of Experimental Zoology A vol 309no 3 pp 166ndash174 2008

6 International Journal of Zoology

[13] M Galgano and C DrsquoAmore ldquoII ciclo dellrsquoepididimo in Lacertas sicula Rafrdquo Italian Journal of Zoology vol 27 no 1-2 pp 1ndash201960

[14] F Angelini and V Botte ldquoSpermatogenesis in reptiles dynamicand regulatory aspectrdquo in Sex Origin and Evolution MucchiSelected Symposia and Monographs UZI R Dallai Ed pp 211ndash230 Mucchi Modena Italy 1992

[15] M Verderame F Angelini and E Limatola ldquoExpression ofestrogen receptor alpha switches off secretory activity in theepididymal channel of the lizard Podarcis siculardquo MolecularReproduction and Development vol 79 no 2 pp 107ndash117 2012

[16] M Verderame and E Limatola ldquoMolecular identification ofestrogen receptors (ER120572 and ER120573) and their differential expres-sion during VTG synthesis in the liver of lizard Podarcis siculardquoGeneral and Comparative Endocrinology vol 168 no 2 pp 231ndash238 2010

[17] V MazziManuale di Tecniche Istologiche e Istochimiche PiccinPadova Italy 1977

[18] M Verderame M Prisco P Andreuccetti F Aniello and ELimatola ldquoExperimentally nonylphenol-polluted diet inducesthe expression of silent genes VTG and ER120572 in the liver of malelizard Podarcis siculardquo Environmental Pollution vol 159 no 5pp 1101ndash1107 2011

[19] MA Younes andCG Pierrepoint ldquoAndrogen steroid-receptorbinding in the canine epididymisrdquoProstate vol 2 no 2 pp 133ndash142 1981

[20] P S Cooke P Young and G R Cunha ldquoAndrogen recep-tor expression in developing male reproductive organsrdquoEndocrinology vol 128 no 6 pp 2867ndash2873 1991

[21] C E Roselli N B West and R M Brenner ldquoAndrogenreceptor and 5120572-reductase activity in the ductuli efferentes andepididymis of adult rhesus macaquesrdquo Biology of Reproductionvol 44 no 4 pp 739ndash745 1991

[22] H O Goyal F F Bartol A A Wiley and C W NeffldquoImmunolocalization of receptors for androgen and estrogenin male caprine reproductive tissues unique distribution ofestrogen receptors in efferent ductule epitheliumrdquo Biology ofReproduction vol 56 no 1 pp 90ndash101 1997

[23] H Ungefroren R Ivell and S Ergun ldquoRegion-specific expres-sion of the androgen receptor in the human epididymisrdquoMolecular Human Reproduction vol 3 no 11 pp 933ndash940 1997

[24] C A Oliveira G A B Mahecha K Carnes et al ldquoDifferentialhormonal regulation of estrogen receptors ER120572 and ER120573 andandrogen receptor expression in rat efferent ductulesrdquoReproduc-tion vol 128 no 1 pp 73ndash86 2004

[25] L OrsquoHaraMWelsh P T K Saunders and L B Smith ldquoAndro-gen receptor expression in the caput epididymal epithelium isessential for development of the initial segment and epididymalspermatozoa transitrdquo Endocrinology vol 152 no 2 pp 718ndash7292011

[26] M T Patrao E J Silva and M C Avellar ldquoAndrogens and themale reproductive tract an overview of classical roles andcurrent perspectivesrdquo Arquivos Brasileiros de Endocrinologia eMetabologia vol 53 no 8 pp 934ndash945 2009

[27] B A Shanbhag and P J Sharp ldquoImmunocytochemical local-ization of androgen receptor in the comb uropygial glandtestis and epididymis in the domestic chickenrdquo General andComparative Endocrinology vol 101 no 1 pp 76ndash82 1996

[28] Q Zhou R Nie G S Prins P T K Saunders B S Katzenellen-bogen and R A Hess ldquoLocalization of androgen and estrogenreceptors in adult male mouse reproductive tractrdquo Journal ofAndrology vol 23 no 6 pp 870ndash881 2002

[29] R Zaya C Hennick and C A Pearl ldquoIn Vitro expression ofandrogen and estrogen receptors in prepubertal and adult ratepididymisrdquo General and Comparative Endocrinology vol 178no 3 pp 573ndash586 2012

[30] V Panet-Raymond B Gottlieb L K Beitel L Pinsky andM ATrifiro ldquoInteractions between androgen and estrogen receptorsand the effects on their transactivational propertiesrdquoMolecularand Cellular Endocrinology vol 167 no 1-2 pp 139ndash150 2000

[31] B Varriale and T Esposito ldquoThe hamster androgen receptorpromoter a molecular analysisrdquo Journal of Steroid Biochemistryand Molecular Biology vol 94 no 1ndash3 pp 103ndash110 2005

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Page 2: Research Article The Involvement of the Androgen Receptor ...downloads.hindawi.com/journals/ijz/2014/457830.pdfThe Involvement of the Androgen Receptor in the Secretion of the Epididymal

2 International Journal of Zoology

treated in the mating period with estradiol-17120573 (E2) This

expression may be related to the morphophysiology of theepididymal channel during the annual reproductive cycle Tocompare the AR expression to the known ERs fluctuation[15] ISH with ER and ER120573 probes on adjacent section wasperformed

2 Material and Methods

21 Animals Adult males of lizard Podarcis sicula (about75ndash8 cm snout vent) were captured near Naples (Italy) fortwo consecutive years during the mating period (spring-early summer) [119899 = 8] and winter stasis (fall-winter) [119899 =8] kept in terraria at natural temperature and photoperiodand fed ad libitum with larvae of mealworm Some samples(119899 = 6) from the mating period were experimentally treatedintraperitoneally with estradiol-17120573 (168 ng100 120583L) in reptilephysiological solution (NaCl 007) every other day for 2weeks [15] In parallel three males were intraperitoneallyinjected with physiological solution (100 120583L) every other dayfor 2 weeks The animals were killed by decapitation afteranaesthesia in ice and the testes with the attached epididymiswere immediately excised and processed for the plannedanalyses All efforts were made to avoid animal suffering

Authorization to capture the animals for experimentaltreatments was granted by the Italian Ministry of the Envi-ronment (Auth SCN2D20009213)

22 Digoxigenin-Labeled Probes cDNA for AR was obtainedby RT-PCR from the total Podarcis testis RNA and amplifiedwith forward primer 51015840-TGGGCAACCTGAAGATGC-31015840and reverse primer 51015840-ACCCCATGGCGAAAATCAT-31015840designed on the known AR sequences of some reptiles Tri-meresurus flavoviridis (AB5483001) Elaphe quadrivirgata(AB5483011) Anolis carolinensis (AF2232242) Calotesversicolor (AF2753702) Trachemys scripta (DQ8489891)Pseudemys nelsoni (AB3010611) Cnemidophorus uniparens(S799381) Alligator mississippiensis (AB1863562) andLeiolepis reeversii rubritaeniata (AB4903851)

First strand cDNA (3 120583L) was used as template in PCRamplification (final volume 25mL) The PCR thermal settingwas as follows 4 min at 94∘C 38 cycles of 30 s at 94∘C 40 sat 50∘C 1min at 72∘C 7min at 72∘CThe obtained fragmentwas sequenced using the BigDye Terminator v11 CycleSequencing Kit (Applied Biosystems) and run on the ABIPRISM 310 Genetic Analyzer and compared to GeneBankdatabase

The ER120572 and ER120573 probes were obtained as alreadydescribed [15 16]

23 Histology and In Situ Hybridization (ISH) The epi-didymis excised from the testes was fixed in Bouinrsquos fluid[17] dehydrated in alcohol increasing gradation clarified inxylene and embedded in paraffin Sections 7 120583m in thicknesswere obtained with Reichert-Jung 2030 microtome Somesections were stained with Malloryrsquos trichrome modified byGalgano [17]

The ISH was performed on adjacent sections with ARER120572 or ER120573 probes and was carried out as described in theliver and epididymis [15 18] Briefly the dewaxed sectionswere treated with proteinase K (10 120583gmL) at 50∘C for 10minDigoxigenin labelled probes were used at a concentrationof 80 ng100 120583L in hybridization buffer [Tris-HCl 002MpH 75 NaCl 03M EDTA 001M DTT 01M formamide50 Denhardtrsquos 1X tRNA 100 120583gmL ss-DNA 100 120583gmL]overnight at 50∘C in a moist chamber The slides wereincubated with RNasi mix at 37∘C for 30min and in thesame mix without RNasi at 37∘C for 30min washed in 2xSSC for 3min in 01x SSC at 60∘C 15min and in NTP andthen incubated in 2 blocking solution [Roche DiagnosticsMannheim Germany] in maleic acid buffer [01M maleicacid 015M NaCl pH 75] for 1 h The sections were keptovernight at 4∘C with an alkaline phosphatase-conjugatedsheep anti-DIG antibody (Roche Diagnostics) (1 2500) inblocking solution and rinsed in NTP [Tris-HCl 01M pH75 NaCl 015M] buffer for 30 min and in NTM buffer[Tris-HCl 100mM pH 95 MgCl 50mM NaCl 100mM] for30min Finally the sections were kept in the dark at roomtemperature in the colour detection substrate solution nitrob-lue tetrazolium and 5-bromo-4-chloro-31015840-Indolylphosphate(BCIPNBT) as recommended by manufacturerrsquo (Roche) inNTM until appearance of the colour To exclude a cross-linkwith genomicDNA some other adjacent sectionswere treatedwith DNAsi Control sections were obtained by omittingincubation with the probes

3 Results

31 Sequencing of Podarcis AR The product of reverse-trans-criptase-PCR from Podarcis testis gave a single band of about300 bp in size (Figure 1(a)) BLAST analysis of the obtainednucleotide sequence (NCBI data bank) revealed high identity(83minus79) to the AR sequences reported in several reptiles(Figures 1(b) and 1(c)) This partial sequence of PodarcisAR cDNA was annotated in EMBL Gen Bank (Accessionnumber JQ219668)

32 ISH with AR Probe The homologous AR probe gavepositive signals continuously throughout the mating periodandwinter stasis in the efferent ductules in the corpus (Figures2(a) and 2(c)) and in the cauda (one for all Figure 2(b))

The epididymal tract of samples treated with estradiol 17-120573 (E2) in the full mating period showed the expression of AR

in the efferent ductules (data not shown) corpus (Figure 1(d))and cauda (data not shown) as in the natural samples Controlanimals treated with physiological solution showed the sameexpression already described for the untreated males (datanot shown)

The sections incubated by omitting the AR probe gavealways negative results (data not shown)

33 ISH with ERs Probe The ISH results on ERs expressionconfirm what has already been found by us in the epididymisof Podarcis [15] In the mating period the ER120572 expressiondid not occur in the secreting corpus (Figure 3(a)) but was

International Journal of Zoology 3

300bp

(a)

Androgen receptors in reptiles1 300

Podarcis156 457Anolis

83

82

82

81

83

83

82

82

82

79

165 466Leiolepis

Calotes 167 468

Cnemid uni 46 348

Trimeresurus 294 594

Protobothrops 153 455

Elaphe 294 594

Trachemys 49 349

Pseudemys 1517 1816

Pelodiscus 94 394

(b)

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

223

234

360

69

115

230

114

283

294

420

129

175

290

174

343

354

480

189

235

350

234

403

414

540

249

295

410

294

463

474

600

300

355

470

354

Podarcis versus other reptiles163

174

300

10

55

170

54

(c)

Figure 1 Sequencing of androgen receptor (a) Agarose gek of RT-PCR on cDNA of Podarcis testis (b) Similarity of predicted amino acidsequences of many reptiles with Podarcis AR (c) BLAST alignment of the nucleotide sequence of AR in Podarcis compared to other reptilesas Anolis Calotes Trimeresurus Trachemys Leiolepis and Cnemidophorus

evident in the efferent ductules (Figure 3(a)) and cauda (datanot shown) During the winter stasis the ER120572 expressionremained evident in the efferent ductules (Figure 3(b)) andcauda (data not shown) and occurred in the corpus devoidof secretion in this period (Figure 3(b))

The ER120573 probe gave positive signals continuouslythroughout the mating period and winter stasis in all theepididymal compartments (data not shown)

In the animals treated in the mating period with E2

an intense ER120572 expression took place in the corpus andthe secretory activity resulted inhibited the efferent ductulesand cauda were still positive (data not shown) ER120573-mRNAdespite the E

2treatment was unchanged compared with

untreated animals (Figure 3(c))

4 Discussion

The epididymis of the lizard Podarcis sicula is characterizedby a cyclic secretion that occurs in the corpus during themating period when this compartment is characterized by acylindrical epithelium producing a great amount of secretorygranules released into the enlarged lumen where many sper-matozoa are travelling from the rete testis In the winter stasisthe cuboidal epithelium surrounds a small totally emptylumen [15] Our previous results demonstrated that this

cyclic secretory activity may be negatively regulated by theexpression of ER120572 In the lizards treated with E

2during the

mating period the epididymal structure becomes equivalentto that observed during the winter stasis the epithelium isreduced in height and the secretory activity stops [15]

The present findings reveal that in Podarcis the epi-didymis may be also androgen dependent and the expressionof AR is constant during the reproductive cycle and along thewhole epididymal length efferent ductules corpus and cauda

The dependence of the epididymis on androgens isknown [8] and in several mammalian species the presence ofAR has been widely demonstrated by variousmethodologicalapproaches [6 19ndash25] In mouse and rat the AR is expressedin a cell-type-specific manner and the protein expression ishigher in the caput and corpus [9] AR-mRNAand protein arealso present in the human with a falling concentration fromthe caput to the cauda [8] It is also known that the androgenscan autoregulate the expression of AR-mRNA promoting ARprotein stabilization too [26]

Among nonmammalian vertebrates the AR protein wasfound in the epithelial cells lining the efferent ductules and inthe epididymal tubules of cockerel [27]

In rooster and drakes a different sensitivity of the epididy-mal segments to androgen has been suggested [11] In reptilesimmunoreactive AR was recorded only in the epididymis ofgreen turtle Chelonia mydas [12]

4 International Journal of Zoology

(a) (b) (c)

(d) (e)

Figure 2 Changes in themorphology andunchanged expression ofAR in the epididymal channel ofPodarcis in the natural cycle or in samplestreated with E

2during the mating period (a) Mating period positive signal for AR-mRNA in the epithelium of the efferent ductules (uarr) and

corpus (lowast) (b) Staining with Malloryrsquos modified trichrome-mating period note the ciliated cubic cells surrounding the efferent ductules (uarr)and the elongated actively secreting cells of the corpusin which lumen is filled with secretory granules (red) and spermatozoa (dark blu) (c)Mating period positivity for AR-mRNA in the cauda (d) Winter stasis both the efferent ductules (uarr) and the corpus (lowast) are still positive (e)The nonsecretory epithelium of the corpus (lowast) expresses AR-mRNA also in E

2treated males in the mating period (lowast) The bar is 30 120583m

In Podarcis it is now shown that the AR-mRNA iscontinuously expressed in the efferent ductules corpus andcauda regardless of the reproductive moment of this seasonalbreederThese results seem in contrast with the plasma levelsof testosterone that reach the maximum during the matingperiod and falls down at the end [14] Since the endotesticularprofile of testosterone shows significant level at the end ofmating remaining discrete throughout the reproductive cycle[14] it is possible that the constant presence of AR-mRNAmay be related to the intratesticular level of androgen even ifat different concentration

The simultaneous presence of several receptors in thesame cells raises questions about the receptors cross talkin the male reproductive system The coexistence of theandrogen and estrogen receptors (ER120572 and ER120573) in malegonad has been discussed in mammals [22 24 28 29]and among the nonmammalian vertebrates only in theturtle [12] In mouse the distribution of ER120573 in the epi-didymis was similar to that of AR [28] In rat ER120573 isconstitutively expressed in the efferent ductules while ARand ER120572 are selectively modulated by their own ligand [24]Physical interactions between AR and ER120572 resulting in

the estradiol-induced modulation of AR transcriptionalactivity have been described to indicate receptors interplayThese interactions arise between the C-terminal ER120572 ligand-binding domain and the N-terminal AR transactivationaldomain or with the full-length AR ER120573 did not interactwith AR [30] Furthermore multiple consensus sequences inthe hamster AR promoter region recognizes as transcriptionfactors ER or AR itself [31]

We recently described in Podarcis that the expression ofER120572 stops during the matings when the cells of the corpus areinvolved in its secretory activity and occurs after E

2treatment

that inhibits the secretion ER120573 expression does not changeneither after E

2treatment [15] The present observations

confirming these pathways for the ERs show at same timethe constant expression of AR throughout the reproductivecycle even in the animals treated with E

2 On the basis

of these results it is possible to suggest that in PodarcisAR expression induces the secretion in the cells lining thecorpus when the ER120572 expression does not occur Converselyduring the winter stasis and in E

2treated males when the

secretory activity of the corpus is inhibited AR and ER120572 arecoexpressed

International Journal of Zoology 5

(a) (b) (c)

Figure 3 ISH with Er120572 or ER120573 homologous probes on the epididymis (a) Mating period Er120572-mRNA is evident in the epithelium of theefferent ductules (uarr) no expression in the secretory epithelium of the corpus (lowast) Note in the corpus the enlarged lumen (lowast) (b) Winter stasisthe Er120572 expression is evident in the efferent ductules (uarr) and in nonsecreting corpus (lowast) (c) E

2treated males in mating period ER120573-mRNA

is expressed in the corpus (lowast) and in the efferent ductules (uarr) Note the restricted empty lumen of the corpus (lowast) The bar is 30 120583m

5 Conclusions

In conclusion in Podarcis AR is expressed in a constantmanner in the whole epididymal length throughout thereproductive annul cycle while the expression of ER120572 iscyclic in the corpus In particular it is possible to arguethat the secretory activity of the epididymal corpus can bepromoted by AR and inhibited by ER120572 The role of ER120573whose expression does not change neither after E

2treatment

remains Unsolved ER120573 may be constitutively expressed andcould ensure the activity of AR by modulating the expressionof ER120572

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The author is grateful to her tutor and teacher ProfessorErmelinda Limatola for critical revision of the paper

References

[1] M L Meistrich T J Hughes and W R Bruce ldquoAlterationof epididymal sperm transport and maturation in mice byestrogen and testosteronerdquo Nature vol 257 no 5531 pp 145ndash147 1975

[2] H DMoore and J M Bedford ldquoShort-term effects of androgenwithdrawal on the structure of different epithelial cells in therat epididymisrdquo Anatomical Record vol 193 no 2 pp 293ndash3111979

[3] S Carreau ldquoEstrogens-male hormonesrdquo Folia Histochemica etCytobiologica vol 41 no 3 pp 107ndash111 2003

[4] B Robaire S Seenundun M Hamzeh and S Lamour ldquoAndro-genic regulation of novel genes in the epididymisrdquoAsian Journalof Andrology vol 9 no 4 pp 545ndash553 2007

[5] R A Hess D Bunick K-H Lee et al ldquoA role for oestrogensin the male reproductive systemrdquoNature vol 390 no 6659 pp509ndash512 1997

[6] G Pelletier ldquoLocalization of androgen and estrogen receptorsin rat and primate tissuesrdquo Histology and Histopathology vol15 no 4 pp 1261ndash1270 2000

[7] C A Oliveira K Carnes L R Franca and R A Hess ldquoInfer-tility and testicular atrophy in the antiestrogen-treated adultmale ratrdquo Biology of Reproduction vol 65 no 3 pp 913ndash9202001

[8] N Ezer and B Robaire ldquoAndrogenic regulation of the structureand functions of the epididymisrdquo in The Epididymis FromMolecules to ClInical Practice B Robaire and B Hinton Edspp 297ndash316 Kluwer AcademicPlenum Publisher New YorkNY USA 2002

[9] S Yamashita ldquoLocalization of estrogen and androgen receptorsin male reproductive tissues of mice and ratsrdquo AnatomicalRecord A vol 279 no 2 pp 768ndash778 2004

[10] B Robaire and M Hamzeh ldquoAndrogen action in the epi-didymisrdquo Journal of Andrology vol 32 no 6 pp 592ndash599 2011

[11] R A P Dornas A G Oliveira M O Dias G A B Mahechaand C A Oliveira ldquoComparative expression of androgenreceptor in the testis and epididymal region of roosters (Gallusdomesticus) and drakes (Anas platyrhynchos)rdquo General andComparative Endocrinology vol 155 no 3 pp 773ndash779 2008

[12] S Otsuka M Suzuki N Kamezaki et al ldquoGrowth-relatedchanges in histology and immunolocalization of steroid hor-mone receptors in gonads of the immature male green turtle(Chelonia mydas)rdquo Journal of Experimental Zoology A vol 309no 3 pp 166ndash174 2008

6 International Journal of Zoology

[13] M Galgano and C DrsquoAmore ldquoII ciclo dellrsquoepididimo in Lacertas sicula Rafrdquo Italian Journal of Zoology vol 27 no 1-2 pp 1ndash201960

[14] F Angelini and V Botte ldquoSpermatogenesis in reptiles dynamicand regulatory aspectrdquo in Sex Origin and Evolution MucchiSelected Symposia and Monographs UZI R Dallai Ed pp 211ndash230 Mucchi Modena Italy 1992

[15] M Verderame F Angelini and E Limatola ldquoExpression ofestrogen receptor alpha switches off secretory activity in theepididymal channel of the lizard Podarcis siculardquo MolecularReproduction and Development vol 79 no 2 pp 107ndash117 2012

[16] M Verderame and E Limatola ldquoMolecular identification ofestrogen receptors (ER120572 and ER120573) and their differential expres-sion during VTG synthesis in the liver of lizard Podarcis siculardquoGeneral and Comparative Endocrinology vol 168 no 2 pp 231ndash238 2010

[17] V MazziManuale di Tecniche Istologiche e Istochimiche PiccinPadova Italy 1977

[18] M Verderame M Prisco P Andreuccetti F Aniello and ELimatola ldquoExperimentally nonylphenol-polluted diet inducesthe expression of silent genes VTG and ER120572 in the liver of malelizard Podarcis siculardquo Environmental Pollution vol 159 no 5pp 1101ndash1107 2011

[19] MA Younes andCG Pierrepoint ldquoAndrogen steroid-receptorbinding in the canine epididymisrdquoProstate vol 2 no 2 pp 133ndash142 1981

[20] P S Cooke P Young and G R Cunha ldquoAndrogen recep-tor expression in developing male reproductive organsrdquoEndocrinology vol 128 no 6 pp 2867ndash2873 1991

[21] C E Roselli N B West and R M Brenner ldquoAndrogenreceptor and 5120572-reductase activity in the ductuli efferentes andepididymis of adult rhesus macaquesrdquo Biology of Reproductionvol 44 no 4 pp 739ndash745 1991

[22] H O Goyal F F Bartol A A Wiley and C W NeffldquoImmunolocalization of receptors for androgen and estrogenin male caprine reproductive tissues unique distribution ofestrogen receptors in efferent ductule epitheliumrdquo Biology ofReproduction vol 56 no 1 pp 90ndash101 1997

[23] H Ungefroren R Ivell and S Ergun ldquoRegion-specific expres-sion of the androgen receptor in the human epididymisrdquoMolecular Human Reproduction vol 3 no 11 pp 933ndash940 1997

[24] C A Oliveira G A B Mahecha K Carnes et al ldquoDifferentialhormonal regulation of estrogen receptors ER120572 and ER120573 andandrogen receptor expression in rat efferent ductulesrdquoReproduc-tion vol 128 no 1 pp 73ndash86 2004

[25] L OrsquoHaraMWelsh P T K Saunders and L B Smith ldquoAndro-gen receptor expression in the caput epididymal epithelium isessential for development of the initial segment and epididymalspermatozoa transitrdquo Endocrinology vol 152 no 2 pp 718ndash7292011

[26] M T Patrao E J Silva and M C Avellar ldquoAndrogens and themale reproductive tract an overview of classical roles andcurrent perspectivesrdquo Arquivos Brasileiros de Endocrinologia eMetabologia vol 53 no 8 pp 934ndash945 2009

[27] B A Shanbhag and P J Sharp ldquoImmunocytochemical local-ization of androgen receptor in the comb uropygial glandtestis and epididymis in the domestic chickenrdquo General andComparative Endocrinology vol 101 no 1 pp 76ndash82 1996

[28] Q Zhou R Nie G S Prins P T K Saunders B S Katzenellen-bogen and R A Hess ldquoLocalization of androgen and estrogenreceptors in adult male mouse reproductive tractrdquo Journal ofAndrology vol 23 no 6 pp 870ndash881 2002

[29] R Zaya C Hennick and C A Pearl ldquoIn Vitro expression ofandrogen and estrogen receptors in prepubertal and adult ratepididymisrdquo General and Comparative Endocrinology vol 178no 3 pp 573ndash586 2012

[30] V Panet-Raymond B Gottlieb L K Beitel L Pinsky andM ATrifiro ldquoInteractions between androgen and estrogen receptorsand the effects on their transactivational propertiesrdquoMolecularand Cellular Endocrinology vol 167 no 1-2 pp 139ndash150 2000

[31] B Varriale and T Esposito ldquoThe hamster androgen receptorpromoter a molecular analysisrdquo Journal of Steroid Biochemistryand Molecular Biology vol 94 no 1ndash3 pp 103ndash110 2005

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

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International Journal of

Microbiology

Page 3: Research Article The Involvement of the Androgen Receptor ...downloads.hindawi.com/journals/ijz/2014/457830.pdfThe Involvement of the Androgen Receptor in the Secretion of the Epididymal

International Journal of Zoology 3

300bp

(a)

Androgen receptors in reptiles1 300

Podarcis156 457Anolis

83

82

82

81

83

83

82

82

82

79

165 466Leiolepis

Calotes 167 468

Cnemid uni 46 348

Trimeresurus 294 594

Protobothrops 153 455

Elaphe 294 594

Trachemys 49 349

Pseudemys 1517 1816

Pelodiscus 94 394

(b)

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

AnolisCalotes

TrimeresurusPodarcis

TrachemysLeiolepis

Cnemidophorus

223

234

360

69

115

230

114

283

294

420

129

175

290

174

343

354

480

189

235

350

234

403

414

540

249

295

410

294

463

474

600

300

355

470

354

Podarcis versus other reptiles163

174

300

10

55

170

54

(c)

Figure 1 Sequencing of androgen receptor (a) Agarose gek of RT-PCR on cDNA of Podarcis testis (b) Similarity of predicted amino acidsequences of many reptiles with Podarcis AR (c) BLAST alignment of the nucleotide sequence of AR in Podarcis compared to other reptilesas Anolis Calotes Trimeresurus Trachemys Leiolepis and Cnemidophorus

evident in the efferent ductules (Figure 3(a)) and cauda (datanot shown) During the winter stasis the ER120572 expressionremained evident in the efferent ductules (Figure 3(b)) andcauda (data not shown) and occurred in the corpus devoidof secretion in this period (Figure 3(b))

The ER120573 probe gave positive signals continuouslythroughout the mating period and winter stasis in all theepididymal compartments (data not shown)

In the animals treated in the mating period with E2

an intense ER120572 expression took place in the corpus andthe secretory activity resulted inhibited the efferent ductulesand cauda were still positive (data not shown) ER120573-mRNAdespite the E

2treatment was unchanged compared with

untreated animals (Figure 3(c))

4 Discussion

The epididymis of the lizard Podarcis sicula is characterizedby a cyclic secretion that occurs in the corpus during themating period when this compartment is characterized by acylindrical epithelium producing a great amount of secretorygranules released into the enlarged lumen where many sper-matozoa are travelling from the rete testis In the winter stasisthe cuboidal epithelium surrounds a small totally emptylumen [15] Our previous results demonstrated that this

cyclic secretory activity may be negatively regulated by theexpression of ER120572 In the lizards treated with E

2during the

mating period the epididymal structure becomes equivalentto that observed during the winter stasis the epithelium isreduced in height and the secretory activity stops [15]

The present findings reveal that in Podarcis the epi-didymis may be also androgen dependent and the expressionof AR is constant during the reproductive cycle and along thewhole epididymal length efferent ductules corpus and cauda

The dependence of the epididymis on androgens isknown [8] and in several mammalian species the presence ofAR has been widely demonstrated by variousmethodologicalapproaches [6 19ndash25] In mouse and rat the AR is expressedin a cell-type-specific manner and the protein expression ishigher in the caput and corpus [9] AR-mRNAand protein arealso present in the human with a falling concentration fromthe caput to the cauda [8] It is also known that the androgenscan autoregulate the expression of AR-mRNA promoting ARprotein stabilization too [26]

Among nonmammalian vertebrates the AR protein wasfound in the epithelial cells lining the efferent ductules and inthe epididymal tubules of cockerel [27]

In rooster and drakes a different sensitivity of the epididy-mal segments to androgen has been suggested [11] In reptilesimmunoreactive AR was recorded only in the epididymis ofgreen turtle Chelonia mydas [12]

4 International Journal of Zoology

(a) (b) (c)

(d) (e)

Figure 2 Changes in themorphology andunchanged expression ofAR in the epididymal channel ofPodarcis in the natural cycle or in samplestreated with E

2during the mating period (a) Mating period positive signal for AR-mRNA in the epithelium of the efferent ductules (uarr) and

corpus (lowast) (b) Staining with Malloryrsquos modified trichrome-mating period note the ciliated cubic cells surrounding the efferent ductules (uarr)and the elongated actively secreting cells of the corpusin which lumen is filled with secretory granules (red) and spermatozoa (dark blu) (c)Mating period positivity for AR-mRNA in the cauda (d) Winter stasis both the efferent ductules (uarr) and the corpus (lowast) are still positive (e)The nonsecretory epithelium of the corpus (lowast) expresses AR-mRNA also in E

2treated males in the mating period (lowast) The bar is 30 120583m

In Podarcis it is now shown that the AR-mRNA iscontinuously expressed in the efferent ductules corpus andcauda regardless of the reproductive moment of this seasonalbreederThese results seem in contrast with the plasma levelsof testosterone that reach the maximum during the matingperiod and falls down at the end [14] Since the endotesticularprofile of testosterone shows significant level at the end ofmating remaining discrete throughout the reproductive cycle[14] it is possible that the constant presence of AR-mRNAmay be related to the intratesticular level of androgen even ifat different concentration

The simultaneous presence of several receptors in thesame cells raises questions about the receptors cross talkin the male reproductive system The coexistence of theandrogen and estrogen receptors (ER120572 and ER120573) in malegonad has been discussed in mammals [22 24 28 29]and among the nonmammalian vertebrates only in theturtle [12] In mouse the distribution of ER120573 in the epi-didymis was similar to that of AR [28] In rat ER120573 isconstitutively expressed in the efferent ductules while ARand ER120572 are selectively modulated by their own ligand [24]Physical interactions between AR and ER120572 resulting in

the estradiol-induced modulation of AR transcriptionalactivity have been described to indicate receptors interplayThese interactions arise between the C-terminal ER120572 ligand-binding domain and the N-terminal AR transactivationaldomain or with the full-length AR ER120573 did not interactwith AR [30] Furthermore multiple consensus sequences inthe hamster AR promoter region recognizes as transcriptionfactors ER or AR itself [31]

We recently described in Podarcis that the expression ofER120572 stops during the matings when the cells of the corpus areinvolved in its secretory activity and occurs after E

2treatment

that inhibits the secretion ER120573 expression does not changeneither after E

2treatment [15] The present observations

confirming these pathways for the ERs show at same timethe constant expression of AR throughout the reproductivecycle even in the animals treated with E

2 On the basis

of these results it is possible to suggest that in PodarcisAR expression induces the secretion in the cells lining thecorpus when the ER120572 expression does not occur Converselyduring the winter stasis and in E

2treated males when the

secretory activity of the corpus is inhibited AR and ER120572 arecoexpressed

International Journal of Zoology 5

(a) (b) (c)

Figure 3 ISH with Er120572 or ER120573 homologous probes on the epididymis (a) Mating period Er120572-mRNA is evident in the epithelium of theefferent ductules (uarr) no expression in the secretory epithelium of the corpus (lowast) Note in the corpus the enlarged lumen (lowast) (b) Winter stasisthe Er120572 expression is evident in the efferent ductules (uarr) and in nonsecreting corpus (lowast) (c) E

2treated males in mating period ER120573-mRNA

is expressed in the corpus (lowast) and in the efferent ductules (uarr) Note the restricted empty lumen of the corpus (lowast) The bar is 30 120583m

5 Conclusions

In conclusion in Podarcis AR is expressed in a constantmanner in the whole epididymal length throughout thereproductive annul cycle while the expression of ER120572 iscyclic in the corpus In particular it is possible to arguethat the secretory activity of the epididymal corpus can bepromoted by AR and inhibited by ER120572 The role of ER120573whose expression does not change neither after E

2treatment

remains Unsolved ER120573 may be constitutively expressed andcould ensure the activity of AR by modulating the expressionof ER120572

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The author is grateful to her tutor and teacher ProfessorErmelinda Limatola for critical revision of the paper

References

[1] M L Meistrich T J Hughes and W R Bruce ldquoAlterationof epididymal sperm transport and maturation in mice byestrogen and testosteronerdquo Nature vol 257 no 5531 pp 145ndash147 1975

[2] H DMoore and J M Bedford ldquoShort-term effects of androgenwithdrawal on the structure of different epithelial cells in therat epididymisrdquo Anatomical Record vol 193 no 2 pp 293ndash3111979

[3] S Carreau ldquoEstrogens-male hormonesrdquo Folia Histochemica etCytobiologica vol 41 no 3 pp 107ndash111 2003

[4] B Robaire S Seenundun M Hamzeh and S Lamour ldquoAndro-genic regulation of novel genes in the epididymisrdquoAsian Journalof Andrology vol 9 no 4 pp 545ndash553 2007

[5] R A Hess D Bunick K-H Lee et al ldquoA role for oestrogensin the male reproductive systemrdquoNature vol 390 no 6659 pp509ndash512 1997

[6] G Pelletier ldquoLocalization of androgen and estrogen receptorsin rat and primate tissuesrdquo Histology and Histopathology vol15 no 4 pp 1261ndash1270 2000

[7] C A Oliveira K Carnes L R Franca and R A Hess ldquoInfer-tility and testicular atrophy in the antiestrogen-treated adultmale ratrdquo Biology of Reproduction vol 65 no 3 pp 913ndash9202001

[8] N Ezer and B Robaire ldquoAndrogenic regulation of the structureand functions of the epididymisrdquo in The Epididymis FromMolecules to ClInical Practice B Robaire and B Hinton Edspp 297ndash316 Kluwer AcademicPlenum Publisher New YorkNY USA 2002

[9] S Yamashita ldquoLocalization of estrogen and androgen receptorsin male reproductive tissues of mice and ratsrdquo AnatomicalRecord A vol 279 no 2 pp 768ndash778 2004

[10] B Robaire and M Hamzeh ldquoAndrogen action in the epi-didymisrdquo Journal of Andrology vol 32 no 6 pp 592ndash599 2011

[11] R A P Dornas A G Oliveira M O Dias G A B Mahechaand C A Oliveira ldquoComparative expression of androgenreceptor in the testis and epididymal region of roosters (Gallusdomesticus) and drakes (Anas platyrhynchos)rdquo General andComparative Endocrinology vol 155 no 3 pp 773ndash779 2008

[12] S Otsuka M Suzuki N Kamezaki et al ldquoGrowth-relatedchanges in histology and immunolocalization of steroid hor-mone receptors in gonads of the immature male green turtle(Chelonia mydas)rdquo Journal of Experimental Zoology A vol 309no 3 pp 166ndash174 2008

6 International Journal of Zoology

[13] M Galgano and C DrsquoAmore ldquoII ciclo dellrsquoepididimo in Lacertas sicula Rafrdquo Italian Journal of Zoology vol 27 no 1-2 pp 1ndash201960

[14] F Angelini and V Botte ldquoSpermatogenesis in reptiles dynamicand regulatory aspectrdquo in Sex Origin and Evolution MucchiSelected Symposia and Monographs UZI R Dallai Ed pp 211ndash230 Mucchi Modena Italy 1992

[15] M Verderame F Angelini and E Limatola ldquoExpression ofestrogen receptor alpha switches off secretory activity in theepididymal channel of the lizard Podarcis siculardquo MolecularReproduction and Development vol 79 no 2 pp 107ndash117 2012

[16] M Verderame and E Limatola ldquoMolecular identification ofestrogen receptors (ER120572 and ER120573) and their differential expres-sion during VTG synthesis in the liver of lizard Podarcis siculardquoGeneral and Comparative Endocrinology vol 168 no 2 pp 231ndash238 2010

[17] V MazziManuale di Tecniche Istologiche e Istochimiche PiccinPadova Italy 1977

[18] M Verderame M Prisco P Andreuccetti F Aniello and ELimatola ldquoExperimentally nonylphenol-polluted diet inducesthe expression of silent genes VTG and ER120572 in the liver of malelizard Podarcis siculardquo Environmental Pollution vol 159 no 5pp 1101ndash1107 2011

[19] MA Younes andCG Pierrepoint ldquoAndrogen steroid-receptorbinding in the canine epididymisrdquoProstate vol 2 no 2 pp 133ndash142 1981

[20] P S Cooke P Young and G R Cunha ldquoAndrogen recep-tor expression in developing male reproductive organsrdquoEndocrinology vol 128 no 6 pp 2867ndash2873 1991

[21] C E Roselli N B West and R M Brenner ldquoAndrogenreceptor and 5120572-reductase activity in the ductuli efferentes andepididymis of adult rhesus macaquesrdquo Biology of Reproductionvol 44 no 4 pp 739ndash745 1991

[22] H O Goyal F F Bartol A A Wiley and C W NeffldquoImmunolocalization of receptors for androgen and estrogenin male caprine reproductive tissues unique distribution ofestrogen receptors in efferent ductule epitheliumrdquo Biology ofReproduction vol 56 no 1 pp 90ndash101 1997

[23] H Ungefroren R Ivell and S Ergun ldquoRegion-specific expres-sion of the androgen receptor in the human epididymisrdquoMolecular Human Reproduction vol 3 no 11 pp 933ndash940 1997

[24] C A Oliveira G A B Mahecha K Carnes et al ldquoDifferentialhormonal regulation of estrogen receptors ER120572 and ER120573 andandrogen receptor expression in rat efferent ductulesrdquoReproduc-tion vol 128 no 1 pp 73ndash86 2004

[25] L OrsquoHaraMWelsh P T K Saunders and L B Smith ldquoAndro-gen receptor expression in the caput epididymal epithelium isessential for development of the initial segment and epididymalspermatozoa transitrdquo Endocrinology vol 152 no 2 pp 718ndash7292011

[26] M T Patrao E J Silva and M C Avellar ldquoAndrogens and themale reproductive tract an overview of classical roles andcurrent perspectivesrdquo Arquivos Brasileiros de Endocrinologia eMetabologia vol 53 no 8 pp 934ndash945 2009

[27] B A Shanbhag and P J Sharp ldquoImmunocytochemical local-ization of androgen receptor in the comb uropygial glandtestis and epididymis in the domestic chickenrdquo General andComparative Endocrinology vol 101 no 1 pp 76ndash82 1996

[28] Q Zhou R Nie G S Prins P T K Saunders B S Katzenellen-bogen and R A Hess ldquoLocalization of androgen and estrogenreceptors in adult male mouse reproductive tractrdquo Journal ofAndrology vol 23 no 6 pp 870ndash881 2002

[29] R Zaya C Hennick and C A Pearl ldquoIn Vitro expression ofandrogen and estrogen receptors in prepubertal and adult ratepididymisrdquo General and Comparative Endocrinology vol 178no 3 pp 573ndash586 2012

[30] V Panet-Raymond B Gottlieb L K Beitel L Pinsky andM ATrifiro ldquoInteractions between androgen and estrogen receptorsand the effects on their transactivational propertiesrdquoMolecularand Cellular Endocrinology vol 167 no 1-2 pp 139ndash150 2000

[31] B Varriale and T Esposito ldquoThe hamster androgen receptorpromoter a molecular analysisrdquo Journal of Steroid Biochemistryand Molecular Biology vol 94 no 1ndash3 pp 103ndash110 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 4: Research Article The Involvement of the Androgen Receptor ...downloads.hindawi.com/journals/ijz/2014/457830.pdfThe Involvement of the Androgen Receptor in the Secretion of the Epididymal

4 International Journal of Zoology

(a) (b) (c)

(d) (e)

Figure 2 Changes in themorphology andunchanged expression ofAR in the epididymal channel ofPodarcis in the natural cycle or in samplestreated with E

2during the mating period (a) Mating period positive signal for AR-mRNA in the epithelium of the efferent ductules (uarr) and

corpus (lowast) (b) Staining with Malloryrsquos modified trichrome-mating period note the ciliated cubic cells surrounding the efferent ductules (uarr)and the elongated actively secreting cells of the corpusin which lumen is filled with secretory granules (red) and spermatozoa (dark blu) (c)Mating period positivity for AR-mRNA in the cauda (d) Winter stasis both the efferent ductules (uarr) and the corpus (lowast) are still positive (e)The nonsecretory epithelium of the corpus (lowast) expresses AR-mRNA also in E

2treated males in the mating period (lowast) The bar is 30 120583m

In Podarcis it is now shown that the AR-mRNA iscontinuously expressed in the efferent ductules corpus andcauda regardless of the reproductive moment of this seasonalbreederThese results seem in contrast with the plasma levelsof testosterone that reach the maximum during the matingperiod and falls down at the end [14] Since the endotesticularprofile of testosterone shows significant level at the end ofmating remaining discrete throughout the reproductive cycle[14] it is possible that the constant presence of AR-mRNAmay be related to the intratesticular level of androgen even ifat different concentration

The simultaneous presence of several receptors in thesame cells raises questions about the receptors cross talkin the male reproductive system The coexistence of theandrogen and estrogen receptors (ER120572 and ER120573) in malegonad has been discussed in mammals [22 24 28 29]and among the nonmammalian vertebrates only in theturtle [12] In mouse the distribution of ER120573 in the epi-didymis was similar to that of AR [28] In rat ER120573 isconstitutively expressed in the efferent ductules while ARand ER120572 are selectively modulated by their own ligand [24]Physical interactions between AR and ER120572 resulting in

the estradiol-induced modulation of AR transcriptionalactivity have been described to indicate receptors interplayThese interactions arise between the C-terminal ER120572 ligand-binding domain and the N-terminal AR transactivationaldomain or with the full-length AR ER120573 did not interactwith AR [30] Furthermore multiple consensus sequences inthe hamster AR promoter region recognizes as transcriptionfactors ER or AR itself [31]

We recently described in Podarcis that the expression ofER120572 stops during the matings when the cells of the corpus areinvolved in its secretory activity and occurs after E

2treatment

that inhibits the secretion ER120573 expression does not changeneither after E

2treatment [15] The present observations

confirming these pathways for the ERs show at same timethe constant expression of AR throughout the reproductivecycle even in the animals treated with E

2 On the basis

of these results it is possible to suggest that in PodarcisAR expression induces the secretion in the cells lining thecorpus when the ER120572 expression does not occur Converselyduring the winter stasis and in E

2treated males when the

secretory activity of the corpus is inhibited AR and ER120572 arecoexpressed

International Journal of Zoology 5

(a) (b) (c)

Figure 3 ISH with Er120572 or ER120573 homologous probes on the epididymis (a) Mating period Er120572-mRNA is evident in the epithelium of theefferent ductules (uarr) no expression in the secretory epithelium of the corpus (lowast) Note in the corpus the enlarged lumen (lowast) (b) Winter stasisthe Er120572 expression is evident in the efferent ductules (uarr) and in nonsecreting corpus (lowast) (c) E

2treated males in mating period ER120573-mRNA

is expressed in the corpus (lowast) and in the efferent ductules (uarr) Note the restricted empty lumen of the corpus (lowast) The bar is 30 120583m

5 Conclusions

In conclusion in Podarcis AR is expressed in a constantmanner in the whole epididymal length throughout thereproductive annul cycle while the expression of ER120572 iscyclic in the corpus In particular it is possible to arguethat the secretory activity of the epididymal corpus can bepromoted by AR and inhibited by ER120572 The role of ER120573whose expression does not change neither after E

2treatment

remains Unsolved ER120573 may be constitutively expressed andcould ensure the activity of AR by modulating the expressionof ER120572

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The author is grateful to her tutor and teacher ProfessorErmelinda Limatola for critical revision of the paper

References

[1] M L Meistrich T J Hughes and W R Bruce ldquoAlterationof epididymal sperm transport and maturation in mice byestrogen and testosteronerdquo Nature vol 257 no 5531 pp 145ndash147 1975

[2] H DMoore and J M Bedford ldquoShort-term effects of androgenwithdrawal on the structure of different epithelial cells in therat epididymisrdquo Anatomical Record vol 193 no 2 pp 293ndash3111979

[3] S Carreau ldquoEstrogens-male hormonesrdquo Folia Histochemica etCytobiologica vol 41 no 3 pp 107ndash111 2003

[4] B Robaire S Seenundun M Hamzeh and S Lamour ldquoAndro-genic regulation of novel genes in the epididymisrdquoAsian Journalof Andrology vol 9 no 4 pp 545ndash553 2007

[5] R A Hess D Bunick K-H Lee et al ldquoA role for oestrogensin the male reproductive systemrdquoNature vol 390 no 6659 pp509ndash512 1997

[6] G Pelletier ldquoLocalization of androgen and estrogen receptorsin rat and primate tissuesrdquo Histology and Histopathology vol15 no 4 pp 1261ndash1270 2000

[7] C A Oliveira K Carnes L R Franca and R A Hess ldquoInfer-tility and testicular atrophy in the antiestrogen-treated adultmale ratrdquo Biology of Reproduction vol 65 no 3 pp 913ndash9202001

[8] N Ezer and B Robaire ldquoAndrogenic regulation of the structureand functions of the epididymisrdquo in The Epididymis FromMolecules to ClInical Practice B Robaire and B Hinton Edspp 297ndash316 Kluwer AcademicPlenum Publisher New YorkNY USA 2002

[9] S Yamashita ldquoLocalization of estrogen and androgen receptorsin male reproductive tissues of mice and ratsrdquo AnatomicalRecord A vol 279 no 2 pp 768ndash778 2004

[10] B Robaire and M Hamzeh ldquoAndrogen action in the epi-didymisrdquo Journal of Andrology vol 32 no 6 pp 592ndash599 2011

[11] R A P Dornas A G Oliveira M O Dias G A B Mahechaand C A Oliveira ldquoComparative expression of androgenreceptor in the testis and epididymal region of roosters (Gallusdomesticus) and drakes (Anas platyrhynchos)rdquo General andComparative Endocrinology vol 155 no 3 pp 773ndash779 2008

[12] S Otsuka M Suzuki N Kamezaki et al ldquoGrowth-relatedchanges in histology and immunolocalization of steroid hor-mone receptors in gonads of the immature male green turtle(Chelonia mydas)rdquo Journal of Experimental Zoology A vol 309no 3 pp 166ndash174 2008

6 International Journal of Zoology

[13] M Galgano and C DrsquoAmore ldquoII ciclo dellrsquoepididimo in Lacertas sicula Rafrdquo Italian Journal of Zoology vol 27 no 1-2 pp 1ndash201960

[14] F Angelini and V Botte ldquoSpermatogenesis in reptiles dynamicand regulatory aspectrdquo in Sex Origin and Evolution MucchiSelected Symposia and Monographs UZI R Dallai Ed pp 211ndash230 Mucchi Modena Italy 1992

[15] M Verderame F Angelini and E Limatola ldquoExpression ofestrogen receptor alpha switches off secretory activity in theepididymal channel of the lizard Podarcis siculardquo MolecularReproduction and Development vol 79 no 2 pp 107ndash117 2012

[16] M Verderame and E Limatola ldquoMolecular identification ofestrogen receptors (ER120572 and ER120573) and their differential expres-sion during VTG synthesis in the liver of lizard Podarcis siculardquoGeneral and Comparative Endocrinology vol 168 no 2 pp 231ndash238 2010

[17] V MazziManuale di Tecniche Istologiche e Istochimiche PiccinPadova Italy 1977

[18] M Verderame M Prisco P Andreuccetti F Aniello and ELimatola ldquoExperimentally nonylphenol-polluted diet inducesthe expression of silent genes VTG and ER120572 in the liver of malelizard Podarcis siculardquo Environmental Pollution vol 159 no 5pp 1101ndash1107 2011

[19] MA Younes andCG Pierrepoint ldquoAndrogen steroid-receptorbinding in the canine epididymisrdquoProstate vol 2 no 2 pp 133ndash142 1981

[20] P S Cooke P Young and G R Cunha ldquoAndrogen recep-tor expression in developing male reproductive organsrdquoEndocrinology vol 128 no 6 pp 2867ndash2873 1991

[21] C E Roselli N B West and R M Brenner ldquoAndrogenreceptor and 5120572-reductase activity in the ductuli efferentes andepididymis of adult rhesus macaquesrdquo Biology of Reproductionvol 44 no 4 pp 739ndash745 1991

[22] H O Goyal F F Bartol A A Wiley and C W NeffldquoImmunolocalization of receptors for androgen and estrogenin male caprine reproductive tissues unique distribution ofestrogen receptors in efferent ductule epitheliumrdquo Biology ofReproduction vol 56 no 1 pp 90ndash101 1997

[23] H Ungefroren R Ivell and S Ergun ldquoRegion-specific expres-sion of the androgen receptor in the human epididymisrdquoMolecular Human Reproduction vol 3 no 11 pp 933ndash940 1997

[24] C A Oliveira G A B Mahecha K Carnes et al ldquoDifferentialhormonal regulation of estrogen receptors ER120572 and ER120573 andandrogen receptor expression in rat efferent ductulesrdquoReproduc-tion vol 128 no 1 pp 73ndash86 2004

[25] L OrsquoHaraMWelsh P T K Saunders and L B Smith ldquoAndro-gen receptor expression in the caput epididymal epithelium isessential for development of the initial segment and epididymalspermatozoa transitrdquo Endocrinology vol 152 no 2 pp 718ndash7292011

[26] M T Patrao E J Silva and M C Avellar ldquoAndrogens and themale reproductive tract an overview of classical roles andcurrent perspectivesrdquo Arquivos Brasileiros de Endocrinologia eMetabologia vol 53 no 8 pp 934ndash945 2009

[27] B A Shanbhag and P J Sharp ldquoImmunocytochemical local-ization of androgen receptor in the comb uropygial glandtestis and epididymis in the domestic chickenrdquo General andComparative Endocrinology vol 101 no 1 pp 76ndash82 1996

[28] Q Zhou R Nie G S Prins P T K Saunders B S Katzenellen-bogen and R A Hess ldquoLocalization of androgen and estrogenreceptors in adult male mouse reproductive tractrdquo Journal ofAndrology vol 23 no 6 pp 870ndash881 2002

[29] R Zaya C Hennick and C A Pearl ldquoIn Vitro expression ofandrogen and estrogen receptors in prepubertal and adult ratepididymisrdquo General and Comparative Endocrinology vol 178no 3 pp 573ndash586 2012

[30] V Panet-Raymond B Gottlieb L K Beitel L Pinsky andM ATrifiro ldquoInteractions between androgen and estrogen receptorsand the effects on their transactivational propertiesrdquoMolecularand Cellular Endocrinology vol 167 no 1-2 pp 139ndash150 2000

[31] B Varriale and T Esposito ldquoThe hamster androgen receptorpromoter a molecular analysisrdquo Journal of Steroid Biochemistryand Molecular Biology vol 94 no 1ndash3 pp 103ndash110 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 5: Research Article The Involvement of the Androgen Receptor ...downloads.hindawi.com/journals/ijz/2014/457830.pdfThe Involvement of the Androgen Receptor in the Secretion of the Epididymal

International Journal of Zoology 5

(a) (b) (c)

Figure 3 ISH with Er120572 or ER120573 homologous probes on the epididymis (a) Mating period Er120572-mRNA is evident in the epithelium of theefferent ductules (uarr) no expression in the secretory epithelium of the corpus (lowast) Note in the corpus the enlarged lumen (lowast) (b) Winter stasisthe Er120572 expression is evident in the efferent ductules (uarr) and in nonsecreting corpus (lowast) (c) E

2treated males in mating period ER120573-mRNA

is expressed in the corpus (lowast) and in the efferent ductules (uarr) Note the restricted empty lumen of the corpus (lowast) The bar is 30 120583m

5 Conclusions

In conclusion in Podarcis AR is expressed in a constantmanner in the whole epididymal length throughout thereproductive annul cycle while the expression of ER120572 iscyclic in the corpus In particular it is possible to arguethat the secretory activity of the epididymal corpus can bepromoted by AR and inhibited by ER120572 The role of ER120573whose expression does not change neither after E

2treatment

remains Unsolved ER120573 may be constitutively expressed andcould ensure the activity of AR by modulating the expressionof ER120572

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The author is grateful to her tutor and teacher ProfessorErmelinda Limatola for critical revision of the paper

References

[1] M L Meistrich T J Hughes and W R Bruce ldquoAlterationof epididymal sperm transport and maturation in mice byestrogen and testosteronerdquo Nature vol 257 no 5531 pp 145ndash147 1975

[2] H DMoore and J M Bedford ldquoShort-term effects of androgenwithdrawal on the structure of different epithelial cells in therat epididymisrdquo Anatomical Record vol 193 no 2 pp 293ndash3111979

[3] S Carreau ldquoEstrogens-male hormonesrdquo Folia Histochemica etCytobiologica vol 41 no 3 pp 107ndash111 2003

[4] B Robaire S Seenundun M Hamzeh and S Lamour ldquoAndro-genic regulation of novel genes in the epididymisrdquoAsian Journalof Andrology vol 9 no 4 pp 545ndash553 2007

[5] R A Hess D Bunick K-H Lee et al ldquoA role for oestrogensin the male reproductive systemrdquoNature vol 390 no 6659 pp509ndash512 1997

[6] G Pelletier ldquoLocalization of androgen and estrogen receptorsin rat and primate tissuesrdquo Histology and Histopathology vol15 no 4 pp 1261ndash1270 2000

[7] C A Oliveira K Carnes L R Franca and R A Hess ldquoInfer-tility and testicular atrophy in the antiestrogen-treated adultmale ratrdquo Biology of Reproduction vol 65 no 3 pp 913ndash9202001

[8] N Ezer and B Robaire ldquoAndrogenic regulation of the structureand functions of the epididymisrdquo in The Epididymis FromMolecules to ClInical Practice B Robaire and B Hinton Edspp 297ndash316 Kluwer AcademicPlenum Publisher New YorkNY USA 2002

[9] S Yamashita ldquoLocalization of estrogen and androgen receptorsin male reproductive tissues of mice and ratsrdquo AnatomicalRecord A vol 279 no 2 pp 768ndash778 2004

[10] B Robaire and M Hamzeh ldquoAndrogen action in the epi-didymisrdquo Journal of Andrology vol 32 no 6 pp 592ndash599 2011

[11] R A P Dornas A G Oliveira M O Dias G A B Mahechaand C A Oliveira ldquoComparative expression of androgenreceptor in the testis and epididymal region of roosters (Gallusdomesticus) and drakes (Anas platyrhynchos)rdquo General andComparative Endocrinology vol 155 no 3 pp 773ndash779 2008

[12] S Otsuka M Suzuki N Kamezaki et al ldquoGrowth-relatedchanges in histology and immunolocalization of steroid hor-mone receptors in gonads of the immature male green turtle(Chelonia mydas)rdquo Journal of Experimental Zoology A vol 309no 3 pp 166ndash174 2008

6 International Journal of Zoology

[13] M Galgano and C DrsquoAmore ldquoII ciclo dellrsquoepididimo in Lacertas sicula Rafrdquo Italian Journal of Zoology vol 27 no 1-2 pp 1ndash201960

[14] F Angelini and V Botte ldquoSpermatogenesis in reptiles dynamicand regulatory aspectrdquo in Sex Origin and Evolution MucchiSelected Symposia and Monographs UZI R Dallai Ed pp 211ndash230 Mucchi Modena Italy 1992

[15] M Verderame F Angelini and E Limatola ldquoExpression ofestrogen receptor alpha switches off secretory activity in theepididymal channel of the lizard Podarcis siculardquo MolecularReproduction and Development vol 79 no 2 pp 107ndash117 2012

[16] M Verderame and E Limatola ldquoMolecular identification ofestrogen receptors (ER120572 and ER120573) and their differential expres-sion during VTG synthesis in the liver of lizard Podarcis siculardquoGeneral and Comparative Endocrinology vol 168 no 2 pp 231ndash238 2010

[17] V MazziManuale di Tecniche Istologiche e Istochimiche PiccinPadova Italy 1977

[18] M Verderame M Prisco P Andreuccetti F Aniello and ELimatola ldquoExperimentally nonylphenol-polluted diet inducesthe expression of silent genes VTG and ER120572 in the liver of malelizard Podarcis siculardquo Environmental Pollution vol 159 no 5pp 1101ndash1107 2011

[19] MA Younes andCG Pierrepoint ldquoAndrogen steroid-receptorbinding in the canine epididymisrdquoProstate vol 2 no 2 pp 133ndash142 1981

[20] P S Cooke P Young and G R Cunha ldquoAndrogen recep-tor expression in developing male reproductive organsrdquoEndocrinology vol 128 no 6 pp 2867ndash2873 1991

[21] C E Roselli N B West and R M Brenner ldquoAndrogenreceptor and 5120572-reductase activity in the ductuli efferentes andepididymis of adult rhesus macaquesrdquo Biology of Reproductionvol 44 no 4 pp 739ndash745 1991

[22] H O Goyal F F Bartol A A Wiley and C W NeffldquoImmunolocalization of receptors for androgen and estrogenin male caprine reproductive tissues unique distribution ofestrogen receptors in efferent ductule epitheliumrdquo Biology ofReproduction vol 56 no 1 pp 90ndash101 1997

[23] H Ungefroren R Ivell and S Ergun ldquoRegion-specific expres-sion of the androgen receptor in the human epididymisrdquoMolecular Human Reproduction vol 3 no 11 pp 933ndash940 1997

[24] C A Oliveira G A B Mahecha K Carnes et al ldquoDifferentialhormonal regulation of estrogen receptors ER120572 and ER120573 andandrogen receptor expression in rat efferent ductulesrdquoReproduc-tion vol 128 no 1 pp 73ndash86 2004

[25] L OrsquoHaraMWelsh P T K Saunders and L B Smith ldquoAndro-gen receptor expression in the caput epididymal epithelium isessential for development of the initial segment and epididymalspermatozoa transitrdquo Endocrinology vol 152 no 2 pp 718ndash7292011

[26] M T Patrao E J Silva and M C Avellar ldquoAndrogens and themale reproductive tract an overview of classical roles andcurrent perspectivesrdquo Arquivos Brasileiros de Endocrinologia eMetabologia vol 53 no 8 pp 934ndash945 2009

[27] B A Shanbhag and P J Sharp ldquoImmunocytochemical local-ization of androgen receptor in the comb uropygial glandtestis and epididymis in the domestic chickenrdquo General andComparative Endocrinology vol 101 no 1 pp 76ndash82 1996

[28] Q Zhou R Nie G S Prins P T K Saunders B S Katzenellen-bogen and R A Hess ldquoLocalization of androgen and estrogenreceptors in adult male mouse reproductive tractrdquo Journal ofAndrology vol 23 no 6 pp 870ndash881 2002

[29] R Zaya C Hennick and C A Pearl ldquoIn Vitro expression ofandrogen and estrogen receptors in prepubertal and adult ratepididymisrdquo General and Comparative Endocrinology vol 178no 3 pp 573ndash586 2012

[30] V Panet-Raymond B Gottlieb L K Beitel L Pinsky andM ATrifiro ldquoInteractions between androgen and estrogen receptorsand the effects on their transactivational propertiesrdquoMolecularand Cellular Endocrinology vol 167 no 1-2 pp 139ndash150 2000

[31] B Varriale and T Esposito ldquoThe hamster androgen receptorpromoter a molecular analysisrdquo Journal of Steroid Biochemistryand Molecular Biology vol 94 no 1ndash3 pp 103ndash110 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 6: Research Article The Involvement of the Androgen Receptor ...downloads.hindawi.com/journals/ijz/2014/457830.pdfThe Involvement of the Androgen Receptor in the Secretion of the Epididymal

6 International Journal of Zoology

[13] M Galgano and C DrsquoAmore ldquoII ciclo dellrsquoepididimo in Lacertas sicula Rafrdquo Italian Journal of Zoology vol 27 no 1-2 pp 1ndash201960

[14] F Angelini and V Botte ldquoSpermatogenesis in reptiles dynamicand regulatory aspectrdquo in Sex Origin and Evolution MucchiSelected Symposia and Monographs UZI R Dallai Ed pp 211ndash230 Mucchi Modena Italy 1992

[15] M Verderame F Angelini and E Limatola ldquoExpression ofestrogen receptor alpha switches off secretory activity in theepididymal channel of the lizard Podarcis siculardquo MolecularReproduction and Development vol 79 no 2 pp 107ndash117 2012

[16] M Verderame and E Limatola ldquoMolecular identification ofestrogen receptors (ER120572 and ER120573) and their differential expres-sion during VTG synthesis in the liver of lizard Podarcis siculardquoGeneral and Comparative Endocrinology vol 168 no 2 pp 231ndash238 2010

[17] V MazziManuale di Tecniche Istologiche e Istochimiche PiccinPadova Italy 1977

[18] M Verderame M Prisco P Andreuccetti F Aniello and ELimatola ldquoExperimentally nonylphenol-polluted diet inducesthe expression of silent genes VTG and ER120572 in the liver of malelizard Podarcis siculardquo Environmental Pollution vol 159 no 5pp 1101ndash1107 2011

[19] MA Younes andCG Pierrepoint ldquoAndrogen steroid-receptorbinding in the canine epididymisrdquoProstate vol 2 no 2 pp 133ndash142 1981

[20] P S Cooke P Young and G R Cunha ldquoAndrogen recep-tor expression in developing male reproductive organsrdquoEndocrinology vol 128 no 6 pp 2867ndash2873 1991

[21] C E Roselli N B West and R M Brenner ldquoAndrogenreceptor and 5120572-reductase activity in the ductuli efferentes andepididymis of adult rhesus macaquesrdquo Biology of Reproductionvol 44 no 4 pp 739ndash745 1991

[22] H O Goyal F F Bartol A A Wiley and C W NeffldquoImmunolocalization of receptors for androgen and estrogenin male caprine reproductive tissues unique distribution ofestrogen receptors in efferent ductule epitheliumrdquo Biology ofReproduction vol 56 no 1 pp 90ndash101 1997

[23] H Ungefroren R Ivell and S Ergun ldquoRegion-specific expres-sion of the androgen receptor in the human epididymisrdquoMolecular Human Reproduction vol 3 no 11 pp 933ndash940 1997

[24] C A Oliveira G A B Mahecha K Carnes et al ldquoDifferentialhormonal regulation of estrogen receptors ER120572 and ER120573 andandrogen receptor expression in rat efferent ductulesrdquoReproduc-tion vol 128 no 1 pp 73ndash86 2004

[25] L OrsquoHaraMWelsh P T K Saunders and L B Smith ldquoAndro-gen receptor expression in the caput epididymal epithelium isessential for development of the initial segment and epididymalspermatozoa transitrdquo Endocrinology vol 152 no 2 pp 718ndash7292011

[26] M T Patrao E J Silva and M C Avellar ldquoAndrogens and themale reproductive tract an overview of classical roles andcurrent perspectivesrdquo Arquivos Brasileiros de Endocrinologia eMetabologia vol 53 no 8 pp 934ndash945 2009

[27] B A Shanbhag and P J Sharp ldquoImmunocytochemical local-ization of androgen receptor in the comb uropygial glandtestis and epididymis in the domestic chickenrdquo General andComparative Endocrinology vol 101 no 1 pp 76ndash82 1996

[28] Q Zhou R Nie G S Prins P T K Saunders B S Katzenellen-bogen and R A Hess ldquoLocalization of androgen and estrogenreceptors in adult male mouse reproductive tractrdquo Journal ofAndrology vol 23 no 6 pp 870ndash881 2002

[29] R Zaya C Hennick and C A Pearl ldquoIn Vitro expression ofandrogen and estrogen receptors in prepubertal and adult ratepididymisrdquo General and Comparative Endocrinology vol 178no 3 pp 573ndash586 2012

[30] V Panet-Raymond B Gottlieb L K Beitel L Pinsky andM ATrifiro ldquoInteractions between androgen and estrogen receptorsand the effects on their transactivational propertiesrdquoMolecularand Cellular Endocrinology vol 167 no 1-2 pp 139ndash150 2000

[31] B Varriale and T Esposito ldquoThe hamster androgen receptorpromoter a molecular analysisrdquo Journal of Steroid Biochemistryand Molecular Biology vol 94 no 1ndash3 pp 103ndash110 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 7: Research Article The Involvement of the Androgen Receptor ...downloads.hindawi.com/journals/ijz/2014/457830.pdfThe Involvement of the Androgen Receptor in the Secretion of the Epididymal

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology