General and Comparative Endocrinology 143 (2005) 21–32 www.elsevier.com/locate/ygcen 0016-6480/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2005.02.026 Brain aromatase from pejerrey Wsh (Odontesthes bonariensis): cDNA cloning, tissue expression, and immunohistochemical localization Pablo H. Strobl-Mazzulla a , Natalia P. Moncaut b , Gabriela C. López a , Leandro A. Miranda a , Adelino V.M. Canario b , Gustavo M. Somoza a,¤ a Laboratorio de IctioWsiología y Acuicultura, Instituto de Investigaciones Biotecnológicas/Instituto Tecnológico de Chascomús (CONICET-UNSAM), C.C. 164, (B7130IWA) Chascomús, Provincia de Buenos Aires, Argentina b Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal Received 13 December 2004; revised 1 February 2005; accepted 21 February 2005 Available online 18 April 2005 Abstract The brain-type aromatase (CYP19A2) cDNA from pejerrey Odontesthes bonariensis was characterized. Its sequence diVers from the ovarian-derived aromatase (CYP19A1) previously reported for the same species. The cDNA is 2305 bp in length and the deduced protein comprises 501 amino-acids. The percentage of identity was higher when compared to other brain-derived aromatase proteins (85–63%) and lower with ovarian-derived aromatases (64–57%). Pejerrey aromatases share 61% of identity. The tissue expression anal- ysis showed that CYP19A2 was expressed in the kidney, brain, and pituitary gland of both sexes and also in the ovary, but not in the eye, spleen, liver, gill, and testis. Semi-quantitative RT-PCR analysis of diVerent brain areas revealed that CYP19A2 was expressed sig- niWcantly higher in anterior male brain areas than in the corresponding female areas, and also when compared to posterior brain areas from both sexes. An immunological analysis using a polyclonal anti-teleost aromatase showed immunoreactive aromatase cells bor- dering the telencephalic ventricle and a strong signal in the ependymal cells of the preoptic area and the hypothalamus. In the optic tectum immunoreactive aromatase cells were labeled in the ventral wall and in the ependymal layer of the third and fourth ventricle with lateral projections. In the pituitary gland immunoreactive aromatase cells could be found in the rostral and proximal pars distalis. In this gland, aromatase Wbers were also detected in diVerent areas; many of them concentrated around blood vessels. 2005 Elsevier Inc. All rights reserved. Keywords: Brain; Aromatase; Sex steroids; Pejerrey 1. Introduction The conversion of androgens (C19) to estrogens (C18) is catalyzed by an enzyme complex, comprising a microsomal aromatase cytochrome P450, product of the CYP19 gene, and a Xavoprotein NADPH-cytochrome P450 reductase (Simpson et al., 1994). 1 Estrogens are hormones with important roles in many physiological and behavioral processes such as, hepatic vitellogenesis in non-mammalian vertebrates (Hiramatsu et al., 2002; Mommsen and Walsh, 1988), germ cell development (Billard, 1992; Wallace, 1985), temperature-sex determination (Pieau and Dorizzi, 2004; Wibbels et al., 1991), sexual diVerentiation * Corresponding author. Fax: +54 2241 424048. E-mail address: email@example.com (G.M. Somoza). 1 Abbreviations used: Dm, area dorsalis telencephali pars medialis; Dd, area dorsalis telencephali pard dorsalis; Dl, area dorsalis telencephali pars late- ralis; Vd, area ventralis telencephalis pars dorsalis; Vv, area ventralis telencephalis pars ventralis; Vp, area ventralis telencephalis pars postcommisuralis; Npp, nucleus preopticus periventricularis; Npo, nucleus preopticus; Oc, optic chiasma; OTec, optic tectum; Ca, cerebral aqueduct; NDM, nucleus dorso- medialis thalami; MT, midbrain tegmentum; NPPv, nucleus posterioris periventricularis; NPGm, nucleus preglomerulosus pars medialis; C, cerebellum; NDLI, nucleus diVusus lobi inferioris; III, third ventricle; IV, fourth ventricle.
Brain aromatase from pejerrey fish ( Odontesthes bonariensis): cDNA cloning, tissue expression, and immunohistochemical localization
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General and Comparative Endocrinology 143 (2005) 21–32
Brain aromatase from pejerrey Wsh (Odontesthes bonariensis): cDNA cloning, tissue expression, and immunohistochemical localization
Pablo H. Strobl-Mazzulla a, Natalia P. Moncaut b, Gabriela C. López a, Leandro A. Miranda a, Adelino V.M. Canario b, Gustavo M. Somoza a,¤
a Laboratorio de IctioWsiología y Acuicultura, Instituto de Investigaciones Biotecnológicas/Instituto Tecnológico de Chascomús (CONICET-UNSAM), C.C. 164, (B7130IWA) Chascomús, Provincia de Buenos Aires, Argentina
b Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal
Received 13 December 2004; revised 1 February 2005; accepted 21 February 2005Available online 18 April 2005
The brain-type aromatase (CYP19A2) cDNA from pejerrey Odontesthes bonariensis was characterized. Its sequence diVers fromthe ovarian-derived aromatase (CYP19A1) previously reported for the same species. The cDNA is 2305 bp in length and the deducedprotein comprises 501 amino-acids. The percentage of identity was higher when compared to other brain-derived aromatase proteins(85–63%) and lower with ovarian-derived aromatases (64–57%). Pejerrey aromatases share 61% of identity. The tissue expression anal-ysis showed that CYP19A2 was expressed in the kidney, brain, and pituitary gland of both sexes and also in the ovary, but not in theeye, spleen, liver, gill, and testis. Semi-quantitative RT-PCR analysis of diVerent brain areas revealed that CYP19A2 was expressed sig-niWcantly higher in anterior male brain areas than in the corresponding female areas, and also when compared to posterior brain areasfrom both sexes. An immunological analysis using a polyclonal anti-teleost aromatase showed immunoreactive aromatase cells bor-dering the telencephalic ventricle and a strong signal in the ependymal cells of the preoptic area and the hypothalamus. In the optictectum immunoreactive aromatase cells were labeled in the ventral wall and in the ependymal layer of the third and fourth ventriclewith lateral projections. In the pituitary gland immunoreactive aromatase cells could be found in the rostral and proximal pars distalis.In this gland, aromatase Wbers were also detected in diVerent areas; many of them concentrated around blood vessels. 2005 Elsevier Inc. All rights reserved.
Keywords: Brain; Aromatase; Sex steroids; Pejerrey
1. Introduction Estrogens are hormones with important roles in
The conversion of androgens (C19) to estrogens(C18) is catalyzed by an enzyme complex, comprising amicrosomal aromatase cytochrome P450, product of theCYP19 gene, and a Xavoprotein NADPH-cytochromeP450 reductase (Simpson et al., 1994).1
0016-6480/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ygcen.2005.02.026
1 Abbreviations used: Dm, area dorsalis telencephali pars medialis; Dd, arearalis; Vd, area ventralis telencephalis pars dorsalis; Vv, area ventralis telencepNpp, nucleus preopticus periventricularis; Npo, nucleus preopticus; Oc, optic cmedialis thalami; MT, midbrain tegmentum; NPPv, nucleus posterioris perivNDLI, nucleus diVusus lobi inferioris; III, third ventricle; IV, fourth ventricle
many physiological and behavioral processes such as,hepatic vitellogenesis in non-mammalian vertebrates(Hiramatsu et al., 2002; Mommsen and Walsh, 1988),germ cell development (Billard, 1992; Wallace, 1985),temperature-sex determination (Pieau and Dorizzi,2004; Wibbels et al., 1991), sexual diVerentiation
dorsalis telencephali pard dorsalis; Dl, area dorsalis telencephali pars late-halis pars ventralis; Vp, area ventralis telencephalis pars postcommisuralis;hiasma; OTec, optic tectum; Ca, cerebral aqueduct; NDM, nucleus dorso-
entricularis; NPGm, nucleus preglomerulosus pars medialis; C, cerebellum;.
22 P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32
(Afonso et al., 2001; D’Cotta et al., 2001; Kitano et al.,1999), and mammalian gonadal development and brainsexual diVerentiation (Lephart, 1996).
In most vertebrates species studied to date aromataseis expressed in the gonads and the brain, but also in avariety of tissues such as liver, adipose tissue, kidney,digestive tract, skin, and placenta (Simpson et al., 2002).
In humans, the CYP19 gene occurs as a single copyand the control of the tissue-speciWc expression dependson the use of an alternative splice 5�-untranslated exonassociated with tissue-speciWc promoter (Simpson et al.,2002). On the other hand, in pigs, multiple tissue-speciWcaromatase isoforms are encoded by three diVerentCYP19 genes (Graddy et al., 2000). However, in teleostWsh two distinct CYP19 loci codifying for two structur-ally and functionally diVerent aromatase forms havebeen found: CYP19A1 and CYP19A2 (Tchoudakovaand Callard, 1998). These forms are preferentiallyexpressed in the gonads and the brain of diVerent bonyWsh species, respectively (Blazquez and Piferrer, 2004;Chang et al., 1997; Chiang et al., 2001; Dalla Valle et al.,2002a,b; Fukada et al., 1996; Gelinas et al., 1998; Kish-ida and Callard, 2001; Kobayashi et al., 2004; Kwonet al., 2001; Tchoudakova and Callard, 1998; Trant,1994). A phylogenetical analyses show that the brainaromatase forms share higher identity between diVerentspecies than their respective ovarian aromatase (Blaz-quez and Piferrer, 2004).
Teleosts Wsh are peculiar because they have an excep-tionally high aromatase activity, 100–1000 times greaterin the preoptic-hypothalamic area compared to adultmammals (Pasmanik and Callard, 1985). Although theadaptive signiWcance of this high level of brain aroma-tase is not well understood, it could be related with theneuroplasticity and neuronal regeneration capability dis-played by adult teleost Wsh, as already suggested (Clintand Zupanc, 2001; Ekström et al., 2001).
The distribution of aromatase activity in vertebratebrains appear to be conserved, and it is localized in fore-brain areas related with the control of reproduction andsexual behavior (Balthazart and Ball, 1998). Also, thebrain aromatase activity has been shown to be sexuallydimorphic in some teleosts Wsh where males displayhigher activity than females in brain areas related toreproduction (González and Piferrer, 2003; Goto-Kazeto et al., 2004; Melo and Ramsdell, 2001), in agree-ment with previous reports in mammals (Lephart, 1996).
The neuroanatomical distribution of brain aromatasehas also been studied by immunohistochemistry and insitu hybridization in three Wsh species, Porichtys notatus,Oncorhynchus mykiss, and Danio rerio (Forlano et al.,2001; Goto-Kazeto et al., 2004; Menuet et al., 2003). Thebrain aromatase mRNA and protein were consistentlyidentiWed bordering the ventricles in the preoptic areaand the hypothalamus. A strong signal was also detectedin the pituitary gland of O. mykiss (Menuet et al., 2003).
In teleost Wsh, immunoreactive aromatase (ir-arom)was observed to occur mostly in glial cells (Forlanoet al., 2001; Menuet et al., 2003) and some expressionwas also demonstrated in neurons (Menuet et al., 2003).This fact is in contrast to mammalian species where aro-matase expression is restricted to neurons cell bodies andnerve terminals (Abe-Dohmae et al., 1994; Roselli,1995); and only expressed in glial cells after brain injury(Garcia-Segura et al., 1999).
In pejerrey, a species with strong temperature sexdetermination (TSD) (Strüssmann et al., 1997), it hasbeen proposed that the brain and pituitary gland haveimportant roles in this process (Miranda et al., 2001,2003). However, Karube et al. (2004) reported in pejerreythat although ovarian aromatase seems to be implicatedin TSD at extreme temperatures, the results obtained forintermediate temperatures were contradictory, suggest-ing that other factors are important during the thermo-labile sex determination period.
The aim of the present work was to isolate the cDNAencoding the pejerrey brain-derived aromatase and char-acterize the tissue expression in both sexes. Also, theneuroanatomical distribution and expression was stud-ied by immunohistochemistry and semi-quantitativePCR. This is the Wrst step to understand the role ofCYP19A2 in the process of sex diVerentiation inpejerrey.
2. Materials and methods
2.1. Animals, tissue preparation, and RNA extraction
Pejerrey were cultured at the IIB-INTECH aquaticfacilities, in outdoor ponds. At the time of tissue collec-tion, the Wsh were anesthetized with benzocaine andhumanitarianly sacriWced by decapitation in accordancewith the UFAW Handbook on the Care and Manage-ment of Laboratory Animals (http://www.ufaw.org.uk/pubs.htm#Lab) and the IIB-INTECH regulations.
Samples were taken from adult Wsh (120–150 g). Eigh-teen month males and females were sampled duringApril; females were in an early vitellogenesis stage andmales were already spermeating according to Strüss-mann (1989). The eyes, kidney, liver, gill, spleen, brain,pituitary, and gonads, were quickly removed and storedin RNAlater (Sigma) at ¡80 °C until analyzed. TotalRNA was extracted using a commercial product, TRIzolreagent (Life Technologies) and kept at ¡80 °C until use.
2.2. cDNA synthesis and ampliWcation of CYP19A2
Two micrograms of total RNA was reverse tran-scribed to cDNA with SuperScript II RNase H¡ (Invit-rogen, Life Technologies) and oligo(dT)12–18 in a totalvolume of 20 �l.
P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32 23
SpeciWc and consensus primers were used (Table 1).Degenerate consensus primers were designed based onthe nucleotide sequence on highly conserved region ofWsh CYP19A2 cDNAs. SpeciWc primers were then syn-thesized according to the pejerrey CYP19A2 sequence.
To amplify the CYP19A2 cDNA a PCR was per-formed in a Wnal volume of 25 �l containing 2.5 �l of 10£reaction buVer, 2�l of a 50 mM MgCl2, 1 �l of a 10 mMdNTPmix, 50 pmol of each primer (Barom1F andBarom1R), 1.25 U of Taq polymerase (Invitrogen, LifeTechnologies) and 1 �l of brain cDNA. After an initial5 min denaturing step at 95 °C, 35 cycles of ampliWcationwere performed using a cycle proWle as following: 95 °Cfor 30 s, 55 °C for 45 s, and 72 °C for 45 s. After the lastcycle, a Wnal elongation step was performed at 72 °C for10 min. Then, a hemi-nested PCR was made using thesame PCR protocol and a 1:500 dilution of the PCR mixwith the following primers: Barom2F and Barom1R.The resulting PCR product having the expected size(855 pb) was excised using a Concert Rapid Gel Extrac-tion System (Gibco-BRL, Life Technologies) and clonedin a bacterial vector using the pGEM-T Easy Kit(Promega). The plasmidic DNA was sequenced at theIIB-INTECH central sequencing facilities and submittedto FASTA for comparison to known sequences accessi-ble in GenBank/EMBL.
New speciWc primers were then designed (BaromF3and BaromR2, see Table 1) based on the obtainedsequence. The primers were tested in a PCR using pejer-rey brain cDNA as a template and gave rise to a 379 bpfragment. This fragment was cloned and sequenced aspreviously described and then used to screen a pejerreybrain and pituitary cDNA library.
2.3. cDNA library construction and screening
A cDNA library was constructed using the UNI-ZAPXR Vector (Strategene) with reverse-transcribed cDNAfrom brains and pituitary glands of adult pejerrey.Approximately 4–5 �g of poly(A)+ RNA was used usinga UNI-ZAP XR cDNA synthesis kit (Stratagene)according to the supplier’s instructions. Membranes lifts(Hybond NX- Amersham-Pharmacia-Biotech) were per-formed in duplicate in order to avoid false positives.These membranes were subsequently denatured for
3 min in 1.5 M NaCl/0.5 M NaOH, neutralized for 3 minin 0.5 M Tris–HCl (pH 8.0)/1.5 M NaCl and Wnallyrinsed for 3 min in 6£ SSC. Membranes were then air-dried and cross-linked.
Membranes were pre-hybridized for 2 h at 56 °C in ahigh stringency solution containing 1 mM EDTA, 7%SDS, and 0.25 M sodium phosphate (Church-Gilberthybridization solution), and hybridized overnight at thesame temperature using fresh solution and theCYP19A2 probe (379 bp) obtained by PCR ampliWca-tion and randomly labeled with [�-32P]dCTP (RediprimeRandom Labeling Kit, Amersham Biosciences). Strin-gency washes were carried out at 56 °C with 2£ SSC, 1£SSC, and 0.1£ SSC containing 0.1% SDS.
SpeciWc hybridization signals were visualized afterexposure to X-ray Wlms. Two independent positive cloneswere isolated after three sequential rounds from two orig-inal pfu of screening, in vivo excised from the ZAPII vec-tor into pBluescript SK(¡) and fully sequenced.
2.4. Phylogenetical analysis
Previously published sequences were obtained fromthe GenBank database and used for amino acid compar-ison and phylogenetic analyses. The amino acidsequences were aligned using the Clustal V developed byHiggins and Sharp (1989), using de DNASTAR soft-ware package. The aligned sequences were then used tobuild phylogenetic trees by distance (Neighbor-Joiningalgorithm) and maximum parsimony criteria PAUP(Phylogenetic Analysis Using Parsimony). Dasyatissabina (stingray) aromatase was used as an out-group toroot the tree.
2.5. RT-PCR analysis for tissue-speciWc expression of brain aromatase
Total RNA (2 �g) from adult male and female pejer-rey tissues (eye, kidney, liver, spleen, gill, brain, pituitary,and gonads) were reversed transcribed using M-MLV(Promega). PCR analysis for CYP19A2 (BaromF3–BaromR2) and �-actin (BactF–BactR) was carried outusing 1 �l cDNA with the following cycle conditions:5 min denaturing step at 95 °C, 35 cycles of ampliWcationwere performed using a cycle proWle of 95 °C for 30 s, 55
Table 1Consensus and speciWc primers used in RT-PCR experiments
24 P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32
or 61 °C for 30 s for �-actin or CYP19A2, respectively,and 72 °C for 30 s. Then, a last elongation step was per-formed at 72 °C for 5 min. The PCR products were ana-lyzed in 1% agarose gel.
2.6. cRNA probe synthesis and Southern hybridization
The cRNA probe synthesis corresponding to CYP19A2(+670/+1048) and �-actin (+237/+749) were ampliWed byPCR from p-GEM-T plasmid (Promega) previouslysequenced to verify probes speciWcity and orientation. ThePCRs were performed using a speciWc and T7 or SP6 prim-ers. The ampliWed DNAs were resolved on 1.2% agarosegel, puriWed and quantiWed. The cRNA probes were thendigoxigenine-labeled by in vitro transcription with T7 orSP6 polymerase using the DIG RNA Labeling Kit (RocheDiagnostics) according to supplier’s instructions.
Three microliters from a 1/10 dilution of the RT-PCRwas used for each tissue. After subjected to electrophore-sis in a 1.2% agarose gel, the DNA on the gel was denat-uralized by soaking it for 45 min in 1.5 M NaCl/0.5 MNaOH, with gently agitation and neutralized by soaking45 min in 0.5 M Tris–HCl (pH 8.0)/1.5 M NaCl. Finally,the DNA was transferred onto a nylon membrane bycapillarity and cross-linked.
Membranes were pre-hybridized for 1 h at 62 °C in20 ml of the Church-Gilber solution and hybridized over-night at the same temperature using a fresh solution and2�l of the speciWc cRNA probes. Stringency washes werecarried out at room temperature with 1£ SSC/0.1% SDSand twice in 0.1£ SSC/0.1% SDS at 68 °C. Membraneswere incubated with Anti-DIG-AP (Roche) and the sig-nal detected with chemiluminoscent CPD-star (Amer-sham Pharmacia) and visualized by autoradiography.
2.7. Semi-quantitative RT-PCR
Semi-quantitative RT-PCRs were performed forCYP19A2 and �-actin using three samples of total RNA(2 �g) from male and female brain sections. The sampleswere divided in four areas as follows: A (olfactory bulbsand telencephalon); B (preoptic area, hypothalamus, androstral optic tectum); C (cerebellum, caudal optic tectum,and medulla oblongata), and D (pituitary gland). All theRNA samples were treated with DNase I (Origin) andreverse-transcribed using SuperScript II RNase H¡
(Invitrogen, Life Technologies) and oligo(dT)12–18 fol-lowing the manufacturer’s instructions.
Each set of primer was tested in a range from 10 to 40cycles, using 1:1 to 1:50 dilution of a cDNA mix derivedfrom the samples, in order to know the number of cycleswhere the product accumulation was in the linear phaseof the curve. For CYP19A2 PCR primers and conditionswere: Barom3F and Barom2R, annealing temperature61 °C and 24 cycles; this reaction gave a 379 bp PCRproduct. For �-actin: BactF and BactR, anneling temper-
ature 55 °C and 18 cycles giving a 517 bp PCR product.All PCR products for each set of primer were resolvedusing the same 1.2% agarose gel and then stained soakingthe gel in a 1% ethidium bromide solution, in order toavoid diVerences between gels and staining.
2.8. Immunohistochemical analysis
Brains from adult male and female pejerrey were Wxedovernight at 4 °C in 4% paraformaldehyde in 0.1 Mphosphate buVer (PB). Brains were then washed in PB,dehydrated and then embedded in Paraplast (Sigma).
The immunocytochemical analysis was carried outusing the avidin–biotin peroxidase method with a com-mercial kit (Vector, Burlingame, USA). Adjacent sagittaland transversal sections (6 �m) were saturated in 0.5%skim milk powder in PBS (0.1 M, pH 7.4) containing 0.3%Triton X-100, and then incubated with the polyclonalanti-teleost aromatase antiserum (kindly provided by Dr.A.H. Bass, Cornell University, USA). This antiserum wasraised against the consensus peptide (CKLQKVKES-FINESLRFHPVV) of Wsh aromatases (Forlano et al.,2001). It was used in a 1:1000 dilution at 4 °C and incu-bated for 24 h. The speciWcity of this antibody was previ-ously demonstrated by Western blot using microsomalbrain and ovary protein fractions (Strobl-Mazzulla et al.,2003). A 57 kDa band (in accordance to the deduced sizeof CYP19A2) was only in the brain samples. All washeswere performed in PBS (0.1 M, pH 7.4) containing 0.3%Triton X-100 and the Wnal reaction product was visual-ized with a 0.1% 3.3�-diaminobenzidine tetrahydrochlo-ride in PBS containing 0.02% H2O2.
The nomenclature of diVerent brain areas wereadopted from the atlas of forebrain nuclei of the goldWshCarassius auratus (Peter and Gill, 1975).
3.1. Isolation of cDNA encoding pejerrey CYP19A2
Pejerrey CYP19A2 (pjCYP19A2) was cloned using acombination of RT-PCR and a brain–pituitary libraryscreening. The cDNA of pjCYP19A2 containing the com-plete open reading frame is 2305bp in length (Fig. 1). Thissequence includes two putative ATG initiation codons, at157 bp and 169 bp starting from the 5� end. Consideringthe Wrst initiation codon, the open reading frame com-prises 1506 bp and the deduced protein is 501 amino-acidlength. The polyadenylation signal (AATAAA) is locatedat 2278 bp. The predicted molecular weight of the proteinencoded by this gene is 56911 Da.
The amino acid sequence includes a putative mem-brane-spanning region (from 17 to 36 residues), a I-helixregion (287 to 321 residues), the Ozol’s peptide region(346 to 368 residues), the aromatase-speciWc region (377
P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32 25
to 399 residues), and the heme-binding region (427 to 3.2. Phylogenetic analysis
The higher identities were found when pejerrey brainaromatase was compared to other brain-derived aromataseproteins (85–63%) and the lower with ovarian-derivedaromatases (64–57%). The two pejerrey aromatases, brainand ovary-derived forms shared only 60.8% (Karube etal., 2004). The conservation degree is even higherbetween aromatases when the putative functionaldomains are considered: the I-helix region variesbetween 75 and 100%; the Ozol’s peptide region, 65 and83%; the aromatase-speciWc region, 65 and 87% and theheme-binding region, 77 and 100%. However, loweridentities were found in the membrane-spanning region,especially in the ovarian derived form 25 to 40%.
The amino acid sequence deduced from the ORF ofpjCYP19A2 cDNA was compared with the amino acidsequences of known aromatases from 28 diVerent teleostspecies. The stingray Dasyatis sabina aromatase wasused as an out group to root the tree. The resulting treeis composed of two main clades corresponding to thebrain and ovarian forms of aromatase which are presentin most if not all teleosts (Fig. 2).
3.3. CYP19A2 tissue-speciWc expression
RT-PCR followed by Southern analysis of 8 diVer-ent tissues (eye, kidney, liver, spleen, gill, brain, pitui-
Fig. 1. Nucleotide and deduced amino acid sequence encoding the cDNA pejerrey CYP19A2. The putative initiation and stop codons are shown inbold. Roman numbers indicate: the membrane-spanning region (I); I-helix region (II); Ozol’s peptide region (III); aromatase-speciWc region (IV); andheme-binding region (V). Numbers refer to the amino acid and nucleotide position in each line.
26 P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32
tary, and gonads) from adult male and female pejerreywas performed for CYP19A2. The �-actin mRNA wasused as a house-keeping gene. Two individual set of tis-sues samples from Wsh at the same gonadal stage wereused for this analysis. The results show thatpjCYP19A2 is mainly expressed in the kidney, brain,and pituitary gland from both sexes. In the ovary aband showing a weak intensity is also observed in theagarose gels but it gave rise to a visible signal after theSouthern hybridization (Fig. 3).
3.4. Semi-quantitative brain-area expression for CYP19A2
The pjCYP19A2 expression of diVerent brain areasfrom male and female pejerrey was analyzed by semi-quantitative RT-PCR (Fig. 4). The brain samples wereextracted when females were in early vitellogeneticstages and males were already spermiating. All the sam-ples gave a visible band for both CYP19A2 and �-actinin the agarose gel. The densitometric analysis of this gelshows that the pjCYP19A2 expression in male wassigniWcantly (p < 0.01) higher in the A and B brain areasthan in the corresponding female brain areas. Also, a
Fig. 3. RT-PCR (upper panel) and Southern hybridization (lowerpanel) of pjCYP19A2 in the eyes, kidney, liver, spleen, gills, brain,pituitary, and gonads from adult male (A) and female (B) pejerrey.AmpliWcation of �-actin was used as internal control. The analysis rep-resents two independent tissues samples.
Fig. 2. Phylogenetic tree of CYP19A proteins. The tree was constructed using a Clustal V multiple sequence alignment program (DNAstar). Thededuced amino acid sequences were used from: Oreochromis mossambicus B (AF135850); Oreochromis niloticus B (AF306786); Orizyas latipes B(AY319970); Epinephelus akaara B (AY547353); Epinephelus coioides B (AY510712); Dicentrarchus labrax B (AY138522); Halichoeres tenuispiniss B(AY489060); Oncorhynchus mykiss BI (AJ311937); O. mykiss BII (AJ311938); Carassius auratus B (AB009335); Pimephales promelas B (AJ277866);Danio rerio B (AF183908); Ictalurus punctatus B (AF417239); Silurus meridionalis B (AY325907); Odontesthes bonariensis B (AY380061); C. auratusA (AF020704); D. rerio A (AF183906); Paralichthys olivaceus A (AB017182); Hippoglossus hippoglossus A (AJ410171); E. akaara A (AY547354); E.coioides A (AY510711); Monopterus albus A (AY583785); D. labrax A (AJ311177); Sparus aurata A (AF399824); Acanthopagrus schlegelii A(AY273211), O. mossambicus A (AF135851); O. niloticus A (U72071); O. latipes A (D82968); O. bonariensis A (not submitted); H. tenuispinis A(AY489061). P450arom derived from Dasyatis sabina (AF097513) was designed as the out-group.
P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32 27
signiWcantly higher expression of pjCYP19A2 wasshown between male A and B areas with respect to the Cand D male and female areas (p < 0.01). No diVerences
Fig. 4. Semi-quantitative RT-PCR of pjCYP19A2 in diVerent male andfemale brain areas. The brains were divided in: olfactory bulbs and telen-cephalum (A); preoptic area, hypothalamus, and rostral optic tectum (B);cerebellum, caudal optic tectum, and medulla oblongata (C); and pituitarygland (D). Values are means § SE of three individual brain areas. (a)Representative ovarian section. (b) Representative testicle section. One-way ANOVA followed Bonferroni’s multiple comparison test analysiswas performed. DiVerent letter indicates signiWcant diVerences (p < 0.01).
were observed in the pjCYP19A2 expression betweenfemale brain areas.
3.5. Neuroanatomical distribution of immunoreactive P450arom cells and projections
Pejerrey transversal and sagittal brain sections fromadult male and female were used with a polyclonal anti-teleost aromatase.
The immunological analysis showed immunoreactivearomatase (ir-arom) cells bordering the telencephalicventricle (Figs. 5A and B) and a strong signal wasobserved in the ependymal cells located at the level ofthe preoptic area and hypothalamus (Figs. 5C, D and6A). The cells have an ovoid nucleus and have elongatedcytoplasmatic processes (Fig. 6B). Also, a few cells werefound in the nucleus preopticus paraventricularis havingno contact with the ventricle (Fig. 6A). In the optic tec-tum, all cells were labeled in the ventral wall sending theprocess dorsally (Fig. 5E). Section at level of the thirdventricle showed labeled cells in the ependymal layer
Fig. 5. Immunohistochemical detection of CYP19A2 in transversal sections of adult pejerrey brain from forebrain to hindbrain (A–H). (A,B) Ir-aromcells bordering the telencephalic ventricle. (C) Ir-arom cells with long cytoplasmic extensions in the ependymal layer at level of the preoptic area. (D)A higher magniWcation of the same cells. (E) Ir-arom cells in the optic tectum. (F) Section at level of the third ventricle showing ir-arom cells in theependymal layer with lateral projections. (G) Ir-arom cells in the periventricular area of the fourth ventricle at the midbrain level. (H) Few ir-cellsbordering the cerebral aqueduct.
28 P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32
with lateral projections (Fig. 5F). Few cells were labeledin the periventricular area of the fourth ventricle (Fig.5G) and dorsal midbrain bordering the cerebral aque-duct (Fig. 5H). Immunoreactive aromatase cells could befound in the rostral pars distalis and in the proximal parsdistalis of the pituitary gland (Figs. 6C and D), also ir-arom Wbers were detected in diVerent areas of the pitui-tary gland; many of them were concentrated aroundblood vessels (Fig. 6E). No immunoreactive signals weredetected either in ovary or testis.
It is now well known that teleost Wsh have at least twoCYP19 loci encoding for two diVerent P450 aromataseenzymes: the ovary-derived or ovarian type, CYP19A1
and the brain-derived or brain type, CYP19A2. In thisstudy, the complete cDNA encoding for pjCYP19A2,diVering from the previously characterized CYP19A1from the same species was isolated and characterized(Karube et al., 2004).
The nucleotide sequence for pjCYP19A2 comprises2305 bp containing the 1506 bp ORF and 501 amino-acid residues from the deduced protein, a larger tran-script but encoding a smaller protein compared topjCYP19A1 which comprises 1776 bp including the1557 bp ORF and 518 amino acid residues (Karubeet al., 2004). This is in agreement with what has beenobserved for other Wsh species where generally the brain-derived form is either of the same size or bigger than theovary-derived transcript: goldWsh B 3.0 kb (Gelinas et al.,1998) vs. goldWsh A 1.9 kb (Tchoudakova and Callard,1998); tilapia nilotica B 2.6 kb (Kwon et al., 2001) vs.
Fig. 6. Immunohistochemical detection of CYP19A2 in sagittal sections of adult pejerrey brain and pituitary gland (A–E). (A) Ir-arom cells in the pre-optic area. The arrows indicate cells with no contact with the ventricle. (B) A higher magniWcation of ventricle contacting ir-arom cells. (C, D) Ir-aromcells in the pituitary gland. (E) Ir-arom cells and Wbers in the pituitary gland. Arrows indicate ir-arom Wbers and the asterisks show blood vessels.
P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32 29
tilapia nilotica A 2.6 kb (Kwon et al., 2001); zebraWsh B4.4 kb (Kishida and Callard, 2001) vs. zebraWsh A 2.1 kb(Kishida and Callard, 2001); rainbow trout B 3.8 kb(Dalla Valle et al., 2002b) vs. rainbow trout A 2.6 kb(Dalla Valle et al., 2002b).
In the pjCYP19A2, two potential initiation trans-duction ATG sites are located at 157 and 169 bp. How-ever, these two putative initiation codons do notpresent the typical nucleotide consensus sequence(CCA/GCCAUGG) proposed by Kozak (1986) whichincreases the translation eYciency; thus the true initia-tion codon remains uncertain. A similar situation hasbeen already found in the CYP19A cDNAs in someother teleost species (Blazquez and Piferrer, 2004;Chang et al., 1997; Dalla Valle et al., 2002a; Fukadaet al., 1996; Tchoudakova and Callard, 1998; Trant,1994).
The present phylogenetic analysis is basically inagreement with the original analysis reported by Cal-lard and Tchoudakova (1997). Pejerrey brain-type aro-matase shares more homology with other brain formsthan the ovarian-type aromatase variants and evenwith the self ovarian-type counterpart. The existence oftwo paralogous genes is in accordance with the pro-posed genomic duplication occurring early in the tele-ost Wsh linage before the divergence of most teleostspecies (Taylor et al., 2003; Vandepoele et al., 2004).The low percentage identity between the ovarian- andbrain-derived aromatases from the same Wsh speciesrepresents an ancient history as separated genes at leastbefore the emergency of euteleost (Chen et al., 2004;Kishida and Callard, 2001) and each branch of the phy-logentic tree shows a clear separation between theAcanthomorpha and Ostariophysi lineages (Fig. 2).These data together with previous reported data sug-gest that during the teleost evolution, the aromatasegene may have suVered a duplication giving rise to thebrain and ovary-derived forms.
The existence of duplicated genes having the sameenzymatic activity in the teleost lineage may have adap-tive advantages providing new functions, diVerent tissueexpression and diVerent ways of regulation (Gonzálezand Piferrer, 2002; Tong et al., 2001; Zhao et al., 2001).In mammals, the tissue-speciWc expression is regulatedby the usage of diVerent promoters (Carreau et al., 2003;Sebastian and Bulun, 2001).
The tissue expression analysis of pjCYP19A2 showsthat it was mainly expressed in the brain, pituitary gland,and kidney of both sexes. The presence of pjCYP19A2 inthe kidney is not surprising because aromatase activityand CYP19A2 expression was recently demonstrated inthe head kidney of the sea bass Dicentrarchus labrax(Blazquez and Piferrer, 2004; González and Piferrer,2003). Also, the presence of pjCYP19A2 in the kidney ofpejerrey is in agreement with the steroidogenic capabilityof Wsh head kidney (Borg, 1994).
Although not previously observed (Strobl-Mazzullaet al., 2003), a low level of pjCYP19A2 expression couldbe demonstrated in the ovary in the same species. Inpejerrey, Karube et al. (2004) have previously demon-strated that aromatase is also expressed in the ovary andspleen and Strobl-Mazzulla et al. (2003) have demon-strated its expression in the testis and brain. The over-lapped expression of both aromatase forms, CYP19A1and CYP19A2, in brain and gonadal tissues has alsobeen found in other Wsh species such as tilapia nilotica(Kwon et al., 2001), rainbow trout (Dalla Valle et al.,2002b), and zebraWsh (Kishida and Callard, 2001). How-ever, Chiang et al. (2001) could only demonstratedCYP19A2 expression in neural tissues in zebraWsh andTchoudakova and Callard (1998) have only foundCYP19A1 expression in the ovary and CYP19A2 in thegonads and the brain of goldWsh. Furthermore also inthe goldWsh, Gelinas et al. (1998) could not demon-strated CYP19A2 expression in the gonads. It is possiblethat Wsh individual diVerences, ages and the reproductivestatus may explain the diVerent observations.
Aromatase activity is correlated with expression levelof aromatase mRNA (Chang et al., 1997; Gelinas et al.,1998), and consequently it can be taken as an indicatorof estrogen synthesis. The aromatization of circulatingandrogens to estrogens in the brain is a key mechanismrelated with many physiological and behavioral pro-cesses, such as sexual diVerentiation of the nervous sys-tem, male sexual behavior, gonadotropins secretion(Balthazart and Ball, 1998), and brain repair (Garcia-Segura et al., 1999).
In the present study, the semiquantitative RT-PCRanalysis of diVerent brain areas shows that males dis-play a signiWcantly higher level of pjCYP19A2 mRNAexpression in anterior and midbrain areas compared tofemales. Also, statistical diVerences between forebrainand midbrain areas with respect to the pituitary, caudaloptic tectum and medulla oblongata, were found only inmales. These results are consistent with those obtainedin other Wsh species where, the dissection of the braininto its major constitutive parts revealed that aroma-tase activity was concentrated in those areas implicatedin the control of reproduction (González and Piferrer,2003; Melo and Ramsdell, 2001; Pasmanik and Cal-lard, 1985). For example, the higher level of aromataseactivity in the preoptic hypothalamic area is coincidentwith the presence of GnRH-I neurons, responsible forthe control of gonadal maturation in teleosts species(Lethimonier et al., 2004). The higher expression inmale pejerrey is consistent with previous studies in seabass, where aromatase activity was always found to behigher in male compared to female brain areas (Gon-zález and Piferrer, 2003). Although Goto-Kazeto et al.(2004) have found variations in the level of expressionbetween females and males in zebraWsh. In this case,although pejerrey were sampled during early sexual
30 P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32
recrudescence, the diVerences observed between sexes,could also be due to sex-linked seasonal variations ofpjCYP19A2 expression associated with the reproduc-tive status. It was already demonstrated in other Wshspecies that either aromatase activity (González andPiferrer, 2003) or expression (Kazeto et al., 2003) varyaccording the reproductive cycle. In this context, fur-ther research is needed to evaluate the expression ofpjCYP19A2 across the reproductive cycle in pejerreyunder natural conditions in order to know if the diVer-ences are sex related, depend on the reproductive statusor both.
Both the Western blot analysis and the immunohis-tochemistry of brain sections were performed using theantibody generated by Forlano et al. (2001) against aconsensus aromatase sequence. However, the Westernanalysis detected a speciWc band of 57 kDa in a micro-somal brain fraction but not in a microsomal ovarianfraction, suggesting that this antiserum can only recog-nize CYP19A2 in this species (Strobl-Mazzulla et al.,2003). An antigenic analysis with the consensussequence used to raise the antiserum together withpjCYP19A1 and pjCYP19A2 reveals that the substitu-tion in the residue 14 (serine by cysteine) decreases theantigenic index of CYP19A1. This could be the reasonwhy this antiserum can only detect pjCYP19A2. Thisinterpretation is supported by the fact that ir-aromcells were only found in pejerrey brain but neither inthe ovary or testis (data not shown). Also, the sameantibody has already been demonstrated to fail indetecting any signal in zebraWsh and goldWsh brain,may be because of three substitutions in residues 9, 12,and 17 (Menuet et al., 2003) and two substitution inresidues 9 and 12, respectively (Forlano et al., 2001).However, in tilapia, Oreochromis sp., where this regionis conserved, this antiserum was demonstrated to showa good signal in the brain (Forlano et al., 2001). Theuse of a highly speciWc antibody against pjCYPA2 willcontribute to clarify this question.
The immunohistochemical analysis shows thatCYP19A2 immunoreactive cells in pejerrey brain weremainly found in periventricular areas from the telen-cephalon, preoptic area, hypothalamus, and the optictectum. A similar localization of brain type aromataseprotein and mRNA were described in previous works,mainly in the periventricular preoptic area in diVerentteleost species (Forlano et al., 2001; Goto-Kazeto et al.,2004; Menuet et al., 2003). In pejerrey, it is interestingto note that the distribution of aromatase expressingcells in the preoptic area, coincides with the localiza-tion of pjGnRH neurons (GnRH-I in this species), thatproject into the pituitary gland (Miranda et al., 2003;Stefano et al., 2000). In this context, the expression ofpjCYP19A2 in the brain and pituitary gland could cor-relate with the reproductive status, suggesting theinvolvement of this gene in the brain–pituitary–
gonadal axis as already suggested in the channel cat-Wsh, Ictalurus punctatus (Kazeto et al., 2003). The fewanatomical and physiological data reported in teleostWsh species in this respect open an important researcharea already extensively studied in mammalian models(Herbison, 1998).
The ir-arom cells detected around the ventricles inpejerrey have long processes resembling those character-istic of the radial glial cells. In this context, Menuet et al.(2003) and Forlano et al. (2001) have shown that braintype aromatase is mainly expressed by glial cells in twodiVerent Wsh species, but Menuet et al. (2003) have dem-onstrated expression in rainbow trout neurons in culture.In clear contrast to Wsh, aromatase is expressed by neu-rons in mammals and birds (Balthazart and Ball, 1998),and it is only highly expressed in glial cells after braininjury in the vicinity of damaged area (Garcia-Seguraet al., 2003). Also in mammals, aromatase activity reachesthe highest level before birth, concomitant with the criti-cal organization period in brain development, falling atthe end of this process (Lephart, 1996), however, in tele-ost Wsh the high brain aromatase activity levels persisteven during adulthood. This fact has been related withthe continuous neurogenesis, migration, plasticity, andregeneration of brain Wsh (Forlano et al., 2001; Gelinas etal., 1998). Nevertheless, the function of the high aroma-tase expression in Wsh brains is still unclear.
The expression of CYP19A2 mRNA and the presenceof ir-arom cells and Wbers were demonstrated in thepejerrey pituitary gland, mainly in the rostral and proxi-mal pars distalis, in agreement with previously publisheddata showing aromatase activity (González and Piferrer,2003) and expression (Goto-Kazeto et al., 2004; Kazetoet al., 2003; Menuet et al., 2003) in the pituitary gland ofother teleost species. To the best of our knowledge, thepresence of ir-arom Wbers has not been reported previ-ously in the pituitary gland of any Wsh. Interestingly, inpejerrey the ir-arom Wbers are mainly located aroundpituitary blood vessels, maybe related with the captureof circulating androgens or to the discharge of estrogensto general circulation. This observation could be associ-ated with feedback process related with the regulation ofthe reproductive axis already described in mammals,where the aromatization of circulating testosterone toestradiol is the predominant regulator of the gonadotro-pin secretion and also inXuence their responsiveness toGnRH (Hayes et al., 2001; Rochira et al., 2002). Furtherresearch is needed to test this hypothesis using Wshmodels.
In summary in pejerrey, as in other teleosts Wsh, atleast two aromatase forms, brain-derived and ovary-derived form are expressed. The pjCYP19A2 tissueexpression is higher in males when compared to femalesand the neuroanatomical localization of pjCYP19A2overlaps with those brain areas related to reproduction.These tools will be useful for future research related with
P.H. Strobl-Mazzulla et al. / General and Comparative Endocrinology 143 (2005) 21–32 31
the mechanisms that control aromatase expression dur-ing the reproductive cycle and during the period of sexdetermination/diVerentiation in a thermolabile sex deter-mining species as pejerrey.
We would like to thank Dr. A. Bass, Cornell Univer-sity for providing the antiserum and Dr. Guillermo Ortí,University of Nebraska-Lincoln for phylogenetic sug-gestions. This work was supported in part by grants ofANPCYT (Pict 01-12168) to G.M.S; SECYT-GRICESProgram PO/PA02-BI/001 to G.M.S. and A.V.M.C.;from the European Commission QLRT-2000-01465 andQLRT-1999-31365 to A.V.M.C. and by a grant-in-aidfrom the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (#15201003).
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