Copulation modifies AR and ERα mRNA expression in the male rat brain

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  Copulation modifies AR and ERα mRNA expression in the male rat brain
  Copulation modi fi es AR and ER  α  mRNA expression in the male rat brain Alonso Fernández-Guasti ⁎ , Paola Arteaga-López, Edwards Antonio-Cabrera Department of Pharmacobiology, CINVESTAV, Calzada de los Tenorios 235, Col. Granjas Coapa, Del. Tlalpan, México City, D. F. 14330 México a b s t r a c ta r t i c l e i n f o  Article history: Received 22 January 2010Received in revised form 13 August 2010Accepted 23 August 2010 Keywords: Rat male sexual satietyLimbic areasAndrogen- and estrogen alpha-receptormessenger ribonucleic acid In situ  hybridization Background/Aims:  One day after male sexual behavior [one ejaculation or copulation to satiety ( ad libitum copulation during 4 h with the same female)] androgen receptor immunoreactivity (AR-ir) is decreased andestrogen receptor alpha immunoreactivity (ER  α -ir) increased in various brain areas related with its control.Seven days after sexual satiety there was a limited recovery of sexual behavior accompanied by a partialrecuperation in the AR-ir. In this study we evaluated if these changes in AR-ir and ER  α -ir were paralleled byvariations in their respective mRNA. Methods:  Sexually experienced male rats were sacri fi ced at different intervals: immediately, 24 h or sevendays after sexual satiety or 24 h after one ejaculation. The changes in AR and ER  α mRNA were analyzed by  insitu  hybridization using digoxigenine-labeled oligonucleotide probes in the MPOA, LSV and the bed nucleusof the stria terminalis, medial division, anterior (BSTMA). Results:  AR mRNA density was decreased in the MPOA and the LSV immediately and 24 h after oneejaculation or sexual satiety. Seven days after copulating to satiety, there was a recovery of AR mRNA. In theBSTMA the different behavioral conditions did not modify the AR mRNA expression. In the MPOA, LSV andBSTMA the ER  α  mRNA increased after a single ejaculation and at all intervals after sexual satiety. Conclusion:  In some brain areas and after some intervals of sexual activity, the changes in steroid proteinreceptors expression seem to be consequence of parallel changes in the expression of the respective mRNA.© 2010 Elsevier Inc. All rights reserved. 1. Introduction Androgens regulate a wide variety of masculine physiologicalfunctions,includingcopulatorybehavior[1 – 3]and,atleastpartly,thiseffectismediatedviatheirconversiontoestrogens[4].Mostandrogenandestrogenactionsareexertedattheirintracellularreceptors,whichexpression is concentrated in limbic areas involved in the regulationof male sexual behavior, such as the medial preoptic area (MPOA),lateral septum (LS), bed nucleus of the stria terminals (BST),ventromedial hypothalamic nucleus (VMH), medial amygdala(MeA) and arcuate nucleus (Arc) [5 – 9]. The functional relationshipbetween steroid sensitive neurons and male sexual behavior in thesebrain areas arises primarily from co-expression and direct steroidactivation results. Regarding the former, a high percentage of neuronsactivated by sexual behavior (established by c-fos) expressesandrogen receptor (AR), estrogen receptor alpha (ER  α ) or both [6].In relation with the later, direct implant of androgens or estrogens insome of these brain areas facilitates copulatory behavior [5,10 – 12],and inversely, AR blockade in MPOA and VMH effectively inhibitsexual behavior in non-castrated males [2,13]. As aforementioned, there is evidence indicating that androgens promote male sexualactivity via their aromatization to estrogens which act primarily onthe ER  α . Thus aromatase inhibition results in a loss of mating [14,15]and knock-out mice lacking either the aromatase or the ER  α  (but notthe ER  β )-gene present severe de fi cits in mating behavior [16 – 18].In male rats the sexual satiety phenomenon is de fi ned as theinhibition of sexual behavior triggered by repetitive ejaculations [19 – 21]. Recently we reported that in MPOA, VMH, LSV and nucleusaccumbens (Acb) the sexual behavior inhibition was paralleled by areduction in AR-immunoreactivity (AR-ir) [8,21]. However, oneejaculation or copulation to satiety did not modify the expression of AR-irintheBST[8,9,21].Interestingly,thereductioninAR-irtriggeredby sexual behavior was not accompanied by changes in the levels of serum androgens [9,21]. The functional association between thereduction in AR-ir and the inhibition of sexual behavior was studiedby analyzing whether the recovery from sexual satiety was accom-panied by an increase of the AR density. Remarkably, the inhibition of sexual behavior coincided with a decrease in AR-ir in the MPOA 24and 48 h after sexual satiety, while seven days later, when there is apartial recovery of copulatory behavior -evidenced as the capacity of all animals to showat least two ejaculations-, the ARdensity returnedto its basal levels [9]. Further functional evidence included therestoration of copulatory behavior in sexually satiated males by theadministration of a mix of anabolic androgens that induce an over-expression of AR  [22]. As to the ER  α , we recently reported that 24 hafter sexual satiety there was an increase in ER  α  density in the BST,LSV, MeA, MPOA and Acb. A single ejaculation was related to anincrease in ER  α  density only in the BST and MeA. ER  α  density in the Physiology & Behavior 101 (2010) 738 – 745 ⁎  Corresponding author. Tel.: +52 55 5483 2870; fax: +52 55 5483 2863. E-mail address: (A. Fernández-Guasti).0031-9384/$  –  see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.physbeh.2010.08.015 Contents lists available at ScienceDirect Physiology & Behavior  journal homepage:  Arc and VMH and serum estradiol levels remained unchanged 24 hafter one ejaculation or mating to satiety [7].The levels of AR and ER  α  depend upon their synthesis, derivedfrom the translation of their mRNA, and from the activity of theproteasome thatis involvedin theirdegradation [23]. Interestingly,asfor other steroid receptors, the amount of AR and ER  α  also dependupon the concentration of their ligands, since for instance androgenspromote the synthesis and prevent the breakdown of AR  [24,25].Accordingly,castrationresultsinarapidvanishingofARinthecentralnervous system, while androgens supplementation produces anincrease in the levels of AR  [22,26 – 28]. The regulation of theexpression of the ER  α  in the brain seems more complex [29]. Thus,castration increases ER  α  mRNA in the MPOA of young male rats [26].By direct contrast, others reported that estradiol binding in the MPOAwas signi fi cantly lower in castrated compared to intact male rats [30].Treatment of male rats with hormones also affects ER  α  binding andimmunoreactivity. Clancy et al. [10] and Yamada et al. [31] reported that counts of ER  α -ir cells in the MPOA, VMH and Arc weresigni fi cantly lower in estradiol-treated male rat compared to thecontrol, and this reductionwas region speci fi c, as a difference was notdetected in the BST, LS, anteroventral periventricular nucleus orlateral mesencephalic central gray. These data indicate that in themale brain, as in the female [32], other factors interact to control ER  α mRNAorproteinexpressioninadditiontosimplyestradiollevels[33].The aim of the present study was to evaluate the putative relationbetween changes in AR- and ER  α -mRNA levels and the expression of sexual behavior. First we studied whether the observed decrease inAR  [8,9,21] and increase in ER  α  [7] were paralleled by a reduction inAR mRNA and an enhancement in ER  α  mRNA to infer whether thedown- or up-regulation of these receptors — produced by sexualactivity — is transcriptional or translational in nature. Secondly, if such parallelism is observed, would the changes in gonadal steroidreceptors reveal a putative association with the inhibition of sexualbehavior triggered by copulation? That is, if at a maximal level of sexual behavior inhibition, for example, immediately after sexualsatiety (after 4 h of   ad libitum  copulation with a single sexually-receptivefemale),there aremore pronounced changesinthe levelsof AR or ER  α  mRNA than seven days later when a partial recovery fromsexual satiety occurs. In addition, to further explore whether theinhibition of sexual behavior could be caused by additive changes insteroid receptors levels, triggered by successive ejaculations, weaddedagroupofmalesthatcopulatedforasingleejaculationwhichisknown to slightly modify sexual performance 24 h later [20,21]. TheMPOA,LSVandBSTwereselectedbecauseofthefollowingreasons:a)their important role on rat copulatory behavior ( vide supra ); b) thecomparison between the putative changes in the mRNA for AR andER  α  with those reported for their respective proteins, and c) theinclusion of brain areas -MPOA and LSV- that show drastic changes insteroid receptors (AR- and ER  α -ir) after sexual satiety [7,21], and of another  — the BST — where the AR-ir was unmodi fi ed after satiety [21].In addition, the neocortex (anterior cingulated cortex, motor andsomatosensoryareasofthefrontoparietalcortices)servedasacontrolbrain area that does not importantly participate in the neural orneuroendocrine control of masculine sexual behavior. 2. Materials and methods  2.1. Animals Adult male (350 – 400 g) albino Wistar rats were used for allexperiments. Animals were kept four to six per cage, with  ad libitum access to rat chow and tap water, under an inverted light – dark cycle(12 heach,lightsoffat10:00 AM)at22 °C.Ratmanagementwasdoneaccordingto the generalprinciples of NIH publication85-23, 1985. AlltheexperimentswereapprovedbythelocalcommitteeofCINVESTAV for ethics on animal experimentation.  2.2. Training tests After adapting at least for one week to the inverted light – darkcycle, all males were given eight mating training tests twice weekly.All the observations were done 1 h after the onset of darkness andunder dim red light. All males were transferred, one each, tocylindrical observation cages. After a 5 min adaptation period, onereceptivefemalewasplacedwitheachanimal.Femalereceptivitywasinduced by sequential s.c. injections of estradiol benzoate (8 μ  g/rat)followed by progesterone (2 mg/rat) 24 and 4 h before the tests,respectively. The copulatory behavior parameters recorded were:intromission latency, number of mounts and intromissions andejaculation latency. The female was removed after the male displayedone ejaculation or after a 30 min period if the animal did not show anintromission. Only the males that showed ejaculation latenciesshorter than 15 min in at least half of the tests were used for furtherexperiments.A control group of sexually experienced males was sacri fi ced bydecapitation at least one week after the last training test. Theexperimental groups consisted of animals sacri fi ced at differentintervals: immediately, 24 h or seven days after sexual satiety. Sexualsatietywasattainedafterexposingasexuallyexperiencedmalewithasingle receptive female for an  ad libitum  copulating period of 4 h.Previous data from our laboratory [34] showed that in 4 h all malesreach the sexual satiety criterion de fi ned as the complete absence of sexual behaviorfor at least 90 min. In addition a groupof animalswassacri fi ced 24 h after they copulated until one ejaculation. Thesegroups and intervals were chosen since previous reports showedimportant changes in AR and ER  α  established by immunocytochem-istry in these brain areas, and because the inhibition of sexualbehavior triggered by repeated ejaculation is maximal immediatelyafterand24 hfollowingsatiety[21,34],whilesevendayslaterthereisa partial recovery of sexual behavior [9]. One day after a single ejaculation there is a very minor inhibition of sexual performance[20]. The n for the groups was of 4 – 6 animals per receptor subtype.The rats were decapitated and the brain was obtained bydissection and washed in a cold and sterile saline solution and thenstoredat − 70 °Cuntilassayed.Thebrainsweremountedonacryostat( − 17 °C) and cut at 8 μ  m sections that were mounted onto poly L-lysine coated slides (75×50 mm). The brain was cut at the level of  − 0.40 mm anterior to bregma according to the Paxinos and Watsoncerebral atlas [35] to have in the same section the LS, the MPOA andthe BST and at a similar antero-posterior level, within these areas thesubnuclei selected were the lateral septal nucleus, ventral part (LSV)and the bed nucleus of the stria terminalis, medial division, anterior(BSTMA). These subnuclei were selected on the bases of previousreports showing changes in steroid receptor proteins associated withsexualactivity[7 – 9,21]. In thissamesection the neocortexexpressionof the gonadal steroid receptors was visualized.  2.3. In situ hybridizationIn situ  hybridization runs included sections of animals in allbehavioral conditions. Digoxigenine-labeled oligonucleotide probeswere obtained from LTD (; Auckland, New Zealand). For AR mRNA the 48mer antisenseoligonucleotide probe hybridizes to nucleotides 755 – 802 usingGenBank accession number NM_012502. For ER- α  mRNA the 28merantisense oligonucleotide probe hybridizes to nucleotides 1633 – 1661using GenBank accession number NM_012689.1.Antisense probes were labeled using digoxigenine (DIG). We usedacommercialprobehyperlabelledwith fi veDIGmoleculesattachedtothe tail of the oligonucleotide. We followed this technique since it iseasy to detect and has a similar sensitivity than that of otherprocedures [36 – 38]. The use of DIG to label nucleic acids is widelyused in molecular biology experiments. The label is introduced by 739  A. Fernández-Guasti et al. / Physiology & Behavior 101 (2010) 738 – 745  enzymatictailingofthe3 ′ -end[39,40].ThereareseveralreportsusingDIG hybridization techniques which developed with NBT/BCIP, forexamples see [41,42].In preparation for hybridization, the tissue sections were placedinto 4% paraformaldehyde in 1× phosphate buffered saline (PBS) for10 min, and then washed with PBS and subsequently air dried.Hybridization was conducted by placing 50 μ  l of hybridization buffer,containing PolyA (10 mg/ml), ssDNA, tRNA, and the labeled probeonto a brain section and covering the section with a coverslip. Slideswereincubatedat43 °C forARand at27 °Cfor ER  α inaneppendorf  insitu  hybridization adapter. Following the incubation, slides weredipped into 1× SSC (standard saline citrate) with 10 mM dithiothrei-tol (DTT) to remove the coverslips and then washed in 1× SSC-DTT atroomtemperature(RT)for10 min,transferredto1×SSC-DTTat55 °Cfor ARand 37 °Cfor ER- α for 15 min,whichwasrepeatedthree times,transferred to 1× SSC-DTT at RT for 30 min, into RT water for 5 min,and allowed to air dry.  2.4. Detection Slides were incubated overnight at 4 °C with anti DIG-AP 1:200 μ  l(Roche Applied Biosystems) placing 50 μ  l per section. The slides werewashed with maleic acid buffer/tween 20 and incubated overnight at4 °C with NBT/BCIP staining solution. Slides were covered withVectashield mounting medium.Microscopic inspection discarded sections where brain regionswere damaged. Within the LSV, the BSTMA and the MPOA a region of interest was selected as shown in Fig. 2. In this region, grayscalephotomicrographs at an ampli fi cation of 40× were done using adigital camera (Pixera Corporation) and 100 positive cells randomlyselected. Within these 100 cells the semi-quantitative determinationof mRNA, established by the staining intensity (mean gray values)was converted to numerical values using a scale from 0 (black) to 255(white)toobtaintheopticaldensity.ThesoftwareLabWorks4.5(UVPbioimaging systems, Co. USA) was used to calculate the opticaldensity. The parameter that changed between the different experi-mental conditions was the optical density of the  fi eld of 100 neuronswithin the region of interest. Alternate brain sections of each subjectwere used to determine the AR and ER  α mRNA. Determinations weredone bilaterally within the same section and in duplicate in everyother section.  2.5. Statistical analyses The average optical density for 100 neurons, within a region of interest, under each experimental condition was calculated. Thesevalues per group for each brain region were compared using a one-way analysis of variance (ANOVA) followed by the Dunnett'stest using the non copulating sexually experienced group as control.Signi fi cance was established at a p b 0.05. Data are expressed asmeans±standard error.  2.6. In situ hybridization controls Control experiments were conducted to con fi rm speci fi c labelingof the oligonucleotide probe to RNA and the speci fi c sequences for AR and ER  α . To determine that the probes were exclusively binding totheir correspondent mRNAs, a speci fi city control experiment wasconducted by comparing the speci fi c versus the unspeci fi c binding.Competition studies with labeled and excess unlabeled proofsdistinguish between speci fi c from unspeci fi c binding, since only theformer is saturable. A competition study with labeled (AR or ER  α  AS- Fig. 1.  Representative photomicrographs of AR mRNA and ER  α  mRNA  in situ  hybridization controls in male rat brains. Competition study utilizing labeled plus excess of unlabeledprobesforARmRNA(A)andforER  α mRNA(E).Labelingspeci fi citycontrolsoftheoligonucleotideprobeestablishedbyusingasenselabeledprobeforARmRNA(B)andER  α mRNA(F).Tissueintegrityandprocedurecontrolsusingantisensepolyd(T)probe(CandG).Experimentalsamplesofratbraintissueprocessedfor insitu hybridizationusingARmRNAAS-DIG (D) and ER  α  mRNA AS-DIG (H). All photomicrographs were taken at 10×. Fig. 2.  Representative photomicrographs of   in situ  hybridization for ER  α - and AR-mRNA in the lateral septal nucleus, ventral part (LSV, panel A), the medial preoptic area (MPOA,panelB)andthebednucleusofthestriaterminalis,medialdivision,anterior(BSTMA,panelC).Schematicdrawingsweretakenfrom fi gure20correspondingto-0.40 mmanteriortobregmaofthePaxinosandWatsoncerebralatlas[32].Nisslstainingsshowtheregionofinterestwithinthesebrainareasat10×.RepresentativephotomicrographsforARmRNAandfor ER  α mRNA at a 40× ampli fi cation of controls (sexually experienced, SEXP), of males sacri fi ced 24 h after one ejaculation (1Ej-24 h) or immediately (SS-Imm), 24 h (SS-24 h) or7 days (SS-7d) after sexual satiety. 3 V, third ventricle, AC, anterior comisure, OC, optic chiasma.740  A. Fernández-Guasti et al. / Physiology & Behavior 101 (2010) 738 – 745  741  A. Fernández-Guasti et al. / Physiology & Behavior 101 (2010) 738 – 745  DIG) and excess of unlabeled (AR or ER  α  AS) probes was done. Anexcess of 10× molar concentration of the unlabeled antisense probeplus the usual concentration of labeled antisense was employed(Fig. 1A, E). The sense control sequence gives a measure of non-speci fi c binding only due to the chemical properties of the probe.Thus, the labeling speci fi city of the oligonucleotide probe wasestablished using a sense labeled probe for AR and ER  α  at 200 ng/ml (Fig. 1B,F). A thirdcontrol wasperformed to determine the tissue-mRNAqualityandef  fi ciencyof theprotocol,usingantisensepolyd(T)probe at 200 ng/ml (Fig. 1C, G). All the sense and antisense probes,labeled or not labeled, as well as the poly d(T) probe, were purchasedfrom Genedetect. Fig. 1 panels D and H shows representativephotomicrographs of rat brain tissue processed for  in situ  hybridiza-tion using the AR or ER  α  AS-DIG probe following the methodpreviously described. As a  fi nal control, we included a brain area,neocortex that does not importantly participate in the neural orneuroendocrine regulation of masculine sexual behavior. 3. Results Following the present technique, the AR mRNA and ER  α -mRNAexpression and distribution in the rat central nervous system wassimilar to those previously reported using other  in situ  hybridizationprocedures [43 – 45]. The control experiments revealed that theoligonucelotide binding was speci fi c and that the tissue quality and in situ  hybridization procedure was optimal (see Fig. 1). In theneocortex there was a widespread steroid receptors mRNA distribu-tion that did not change after copulation.Fig. 2 shows representative photomicrographs of nissl staining (at10×) showing the regions of interest within the LSV, MPOA andBSTMA (panels A, B and C, respectively). This  fi gure also showsrepresentativephotomicrographs(at40×)forER  α -andAR-mRNAsincontrols and in males sacri fi ced after sexual behavior. Note that in theLSV and MPOA, but not in the BSTMA, there was a drastic decrease inthe level of AR mRNA in the males sacri fi ced immediately after sexualsatiety or 24 h after one ejaculation or copulation to satiety. By dayseven after sexual satiety there was a recovery in the levels of AR mRNAintheLSVandMPOA.FortheER  α mRNAtherewasaconsistentincrease after sexual behavior in all three brain structures.The densitometric analyses of the AR mRNA in the different brainregions after various intervals and amounts of sexual activity areshown in Fig. 3. In the LSV (Fig. 3, upper panel) the AR mRNA expression showed statistical signi fi cant differences (one wayANOVA: F 4,15 =12.618, p b 0.001). This expression in animals thatejaculatedonceandsacri fi ced24 hlateroreuthanizedimmediatelyor24 h after sexual satiety was signi fi cantly diminished compared tothat shown by control animals. Seven days after sexual satiety, therewas a tendency to recover basal levels, although the diminution wasstill statistical signi fi cant when compared to the control group. In theMPOA (Fig. 3, middle panel) the AR mRNA expression followed asimilar pattern to that shown by the LSV and also showed statisticalsigni fi cant differences (one way ANOVA: F 4,13 =23.626, p b 0.001). Inthis brain area there was a decrease in AR mRNA in those animalssacri fi ced 24 h after one ejaculation or euthanized immediately or24 hpost-sexualsatiety.Theanimalssacri fi cedsevendayspostsexualsatiety recovered the levels of AR mRNA expression when comparedwith the control group. In the BSTMA (Fig. 3, lower panel) the AR mRNA expression did not vary between control animals and thosethat copulated regardless of whetherthey achievedoneejaculationorwere sexually satiated (one way ANOVA: F 4,14 =2.241, nonsigni fi cant).Fig. 4 shows the densitometric analyses of the ER  α  mRNA in thedifferent brain regions after various intervals and amounts of sexualactivity. The ER  α  mRNA expression among the different groupsshowed statistical signi fi cant differences in the three brain areasanalyzed (One way ANOVAs for the different brain regions: LSV:F 4,12 =20.521 , p b 0.001; MPOA: F 4,12 =18.315, p b 0.001; BSTMA:F 4,12 =17.672, p b 0.001). Clearly, in all brain areas there was a drasticincrease in the expression of ER  α  mRNA. This increase wasindependent of the amount of sexual behavior and of the sacri fi ceinterval after sexual satiety. 4. Discussion The present results show that immediately after sexual satiety or24 h after one ejaculation or copulation to satiety there is a reductionin AR mRNA and an increase in ER  α mRNA in brain areas related withits control. For both steroid receptors mRNA's these changes wereindependent of the amount of sexual activity that is, the AR mRNAdecrease and ER  α  mRNA increase were similar between malessacri fi ced 24 h after one ejaculation and those euthanized 24 h orimmediately after copulation to satiety. However the reduction in AR mRNAwasregionalandtimeselectivebecausesevendaysaftersexualsatiety there was a recovery in AR mRNA in the LSV and MPOA. In theBSTMA there were no changes in the AR mRNA expression regardlessof the amount of sexual behavior and the timing after sexual satiety.Conversely, the ER  α  mRNA was equally high expressed after oneejaculation and at all intervals after sexual satiety in these three brainareas studied.  BSTMA **    A   R  m   R   N   A  m  e  a  n   O .   D .   (  o  p   t   i  c  a   l   d  e  n  s   i   t  y   ) LSV ***** SEXP SS-24h SS-7d0. ** MPOA **** SS-Imm1EJ-24h Fig. 3.  The  fi gure shows the mean±SE optical density of AR mRNA expression in thelateral septal nucleus, ventral part (LSV), the medial preoptic area (MPOA) and the bednucleus of the stria terminalis, medial division, anterior (BSTMA) of male rats. Theanalyses were done in 100 neurons in the regions of interest as shown in Fig. 2. SEXP: sexual experienced males not allowed to copulate (N=4), 1Ej-24 h: male ratssacri fi ced 24 h after one ejaculation (N=4), SS-Imm: sexually exhausted male ratssacri fi cedimmediatelyafterthesatietytest(N=3),SS-24 h:sexuallysatiatedmaleratssacri fi ced 24 h later (N=4) and SS-7d: sexually satiated male rats sacri fi ced 7 dayslater (N=4). Dunnett t test, * p b 0.05; ** p b 0.02 compared with SEXP.742  A. Fernández-Guasti et al. / Physiology & Behavior 101 (2010) 738 – 745
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