IMMUNOLOCALISATION OF α AND β OESTROGEN RECEPTORS IN BASOLATERAL AMYGDALA OF RABBIT MALES

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1 Bull Vet Inst Pulwy 56, 83-87, 2012 IMMUNOLOCALISATION OF α AND β OESTROGEN RECEPTORS IN BASOLATERAL AMYGDALA OF RABBIT MALES GRZEGORZ LONC Deprtment of Animl Antomy nd Histology, Fulty of Veterinry Mediine, University of Life Sienes, Lulin, Polnd Reeived: My 11, 2011 Aepted: Novemer 8, 2011 Astrt Expression of α (ER-α) nd β (ER-ß) oestrogen reeptors in neurons of solterl omplex of the mygdl ws studied in rit mles. The exmintions were rried out on rins of 12 sexully mture rit mles weighing 3-4 kg. An immunohistohemil method (IHC) with primry Er-α ntiodies, NCL-L6F11 nd Er-ß: NCL-ERet lone EMR02 ws pplied. The perentge of immunoretive neurons ws determined. Morphometri nd sttistil nlysis of immunoretive (ER+) neurons ws rried out inluding the lolistion of IHC retion within the exmined neurons. The min ple of IHC-ER-α retion ws the ytoplsm of nervous ells, wheres in the exmintion of IHC-ER-β, the retion ws oserved minly within ell nuleus. The nulei of solterl omplex re region of reltively high perentge of ER-β+ neurons nd lower perentge of ER-α+ neurons in omprison with other nulei of the mygdl. Bsolterl nuleus ws hrterised y higher perentge of ER- β+ neurons in whih the retion ws oserved in the ytoplsm. Key words: rit, mygdl, oestrogen reeptor, solterl. Oestrogens ffet tissues through inding with speifi reeptors present in trget tissues. Oestrogen reeptors our in two min forms: α nd β (ER-α nd ER-β). They re lignd dependent ftors tivting trnsription. Struturlly within oestrogen reeptors one n distinguish six funtionl suunits lelled from A to F. Oestrogen reeptors α nd β differ from one nother within the domins (6, 20). As demonstrted in experimentl studies oestrogens ply n importnt role not only in the funtioning of the reprodutive system ut hve signifint impt on mny other orgns nd tissues not relted to reprodution, inluding strutures nd funtions of the entrl nervous system in oth femles nd mles. They ffet lso mturtion, prolifertion, nd lifespn of neurons, nd synptogenesis (1, 13). The mygdl (orpus mygdloideum, CM) nd hippompus re the min enters of the limi system (16). Funtioning of these rin enters is primrily ssoited with memory nd emotions. Through efferent juntions it lso ffets utonomi nervous system s funtions. Chnges in neurons of the mydgl nd hippompus ording to mny sientists ompny suh disorders s Alzheimer s disese, shizophreni, depression (8, 12). The mygdl plys n importnt role in the regultion of defensive reflexes in humns nd nimls. It plys ruil role in nxiety retion (9, 10). Within the mygdl one n distinguish phylogenetilly younger solterl omplex (BLC), phylogenetilly older ortiomedil omplex (CMC), nd so-lled other nulei. Into solterl omplex the following nulei re inluded: lterl (LA), solterl (BL), nd somedil (BM). Morphology nd ytorhiteture of the mydgl in rits hve een thoroughly desried in sientifi literture (4, 7, 21). Studies on morphology of the mygdl in oth mles nd femles showed tht it is lso hrterised y sexul dimorphism. Antomil differenes within the mygdl of mles nd femles minly referred to medil nuleus. They onern volume of nerve nulei, neuron size, nd density of dendriti spines (3, 15, 19). Mteril nd Methods The reserh mteril onsisted of the rin olleted from 12 sexully mture rit mles (Orytolgus uniulus) of New Zelnd reed, weighing 3-4 kg. The mteril me from rit slughterhouse. The olleted mteril ws divided into smller frgments nd emedded in prffin loks ording to the rules dopted in immunohistohemil exmintions. The mteril from the right rin hemispheres ws used in the reserh. The histologil slides were prepred from the loks utting the

2 84 hemispheres frontlly into 5 µm thik setions. The setions were pled on silnised glsses SuperFrostRPlus (MENZEL-GLASER). The preprtions were inuted for 48 h t 4 o C with primry monolonl mouse IgG1 Er-α ntiody (NCL- L6F11 lone, Novostr) diluted 1:40 nd monolonl IgG1 Er-β ntiody (NCL-ER-β lone EMR02, Novostr) diluted 1:50. Visulistion ws performed y LAB/LSAB method using system for visulistion LSAB (DAKO). In order to diversify the setions, they were stined with Myer s hemtoxylin. In the ples of positive immunohistohemil retion, finl insolule retion produt of rown olour, inditing the presene of reeptors ws visile. Control setions were inuted without primry ntiody. Finlly, the setions were losed in Cndin lm. The results of immunohistohemil retion were viewed under light mirosope (OLYMPUS BX40). To determine the strutures of the mydgl, tlses nd pulitions desriing the struture nd ytorhiteture of rit s mydgl were used (4, 5, 7, 21). Photogrphi doumenttion ws performed using digitl mer COLORVIEW IIIu (soft Imging System). The otined imges, sved in TIF formt, underwent morphometri nlysis using ell D progrmme (Soft Imging System). The min differentition riterion ws the presene of the retion or its lk, s well s intrellulr lolistion of immunohistohemil retion produt. Sttistil nlysis ws rried out using omputer progrmme Stsisti version 6.0PL. The otined results were nlysed using one-wy nlysis of vrine (ANOVA). Sttistil signifine of differenes etween the nlysed results ws determined using post-ho test of Tukey. Proility vlue P<0.05 ws dopted s the oundry of sttistil signifine of the otined differenes nd orreltions. For eh prmeter rithmeti men (x _ ) ws lulted s well s stndrd devition (SD) nd stndrd error of men SEM); the presented results were shown in the form of x _ ±SD. Results IHC retion for the presene of α nd β oestrogen reeptors ws oserved within neurons oth in the nuleus nd ytoplsm of nervous ells. On the sis of lolistion of the retion, the exmined neurons were divided into four groups: 1. IHC ER retion ws oserved within the ytoplsm of neurons. 2. IHC-ER retion ws oserved within nuleus of neuron. 3. IHC ER retion ws oserved within the ytoplsm s well s nuleus of neuron. 4. lterl nuleus ER-α lterl nuleus ER-β d yt + nu + yt+/nu+ yt-/nu- 39% 6% 21% 33% yt + nu + yt+/nu+ yt-/nu- 1% 69% 8% 22% somedil nuleus ER-α somedil nuleus ER-β yt + nu + yt+/nu+ yt-/nu- 27% 11% 26% 36% yt + nu + yt+/nu + yt-/nu- 3% 75% 7% 15% solterl nuleus ER-α solterl nuleus ER-β yt + nu + yt+/nu+ yt-/nu- 29% 12% 25% 34% yt+ nu+ yt+/nu+ yt-/nu- 4% 58% 19% Fig. 1. Immunohistohemil expression of α nd β oestrogen reeptors in solterl nulei of the mygdl in rit mle s rin. Sttistilly signifint differenes were mrked with different letter symols for the vlue of proility P 0.05.

3 85 Fig. 2. Expression of oestrogen reeptors in immunohistohemil exmintions: 1. ER-α in lterl nuleus, 2. ER-α in somedil nuleus, 3. ER-α in solterl nuleus, 4. ER-ß in lterl nuleus, 5. ER-ß in somedil nuleus, 6. ER-ß in solterl nuleus. Aout 200x No IHC retion for ER ws oserved. In the mjority of the immunoretive neurons in the region of ll exmined nervous nulei, the produt of IHC retion for presene of ER-α reeptors ws oserved in the ytoplsm of nervous ells, wheres the presene of ERβ ws oserved minly within neuron nulei. Detiled results of retion lolistion in neurons of individul nulei of solterl omplex of the mygdl were demonstrted in form of hrts. (Figs 1-6). In nulei of solterl omplex, higher perentge of neurons in whih IHC-ER-β retion ws demonstrted in reltion to other mygdl nulei ws oserved. At the sme time, in reltion to the remining nulei of the omplex, this region ws hrterised y lower perentge of neurons in whih IHC-ER-α retion ws oserved. In individul nulei of the omplex, ER-α expression ws similr, the min retion ple ws the ytoplsm of the exmined neurons. In solterl nuleus there ws signifintly higher perentge of neurons in whih ytoplsmti retion for ER-β ws oserved (Figs 1, 2).

4 86 Disussion In the studies on distriution of β-oestrogen reeptors in the rin of rt mles exposed to ovrietomy, immunoretive neurons of different rin regions were oserved, mong others in mygdl. Within mygdle, the presene of ER- β ws oserved in neurons of medil nuleus nd ortil nulei (23). Expression of β oestrogen reeptors ws lso ompred in femles nd mles. In rt s rin, the regions hrterised y high immunoretivity in oth femles nd mles were distinguished. Retion of reeptors within neurons ws oserved in the ytoplsm nd ell nuleus. The differenes etween mles nd femles referred to suh strutures s the nuleus mmmilris medilis, lous eruleus, nuleus retiulris pontis. In these rin regions the reeptors were seen only in mles. Only in femles β-oestrogen reeptors were present in the nuleus vestiulris superior (24). One lso distinguished rin regions; in whih immunoretive neurons were oserved in oth sexes; however, n inrese in retion in femles nd mles ws different (24). The differenes were lso oserved in intrellulr reeptor s lotion. In the mygdl, the retion only in ell nulei of neurons ws oserved (24). Other studies hve ompred expression of α- nd β-oestrogen reeptors in individuls of the sme gender. The exmintion results hve proved uneven distriution of isoforms of oestrogen reeptor in the rin of ovrietomised mouse nd rts femles (14, 22). The rried out exmintions hve reveled rin regions in whih high perentge of immunoretive neurons of oth isoforms of oestrogen reeptor e.g. ed nuleus of the stri terminlis nd nuleus medilis mygdl ws oserved (14). Additionlly, regions in whih predominted individul isoforms were oserved. In the mygdl of ovrietomised mie, immunohistohemil retion for the presene of oth isoforms of oestrogen reeptor ws demonstrted in ll regions; however, the medil nuleus ws the ple in whih undnt retion for α nd β oestrogen reeptors ws oserved. Stronger retion for the presene of α oestrogen reeptors ws shown in the mygdlohippompl re (14). Immunohistohemil exmintions for the presene of α-oestrogen reeptors were lso rried out in ovrietomised rit femles. In the mygdle, retion of strong density ws oserved in the medil mygdloid nuleus, sl nulei, nd ortil nulei. Wek retion ws lso oserved in the entrl mydgloid nuleus (2). There re no dt on the expression of the exmined reeptors in other regions of the mygdl. In the sme exmintions, the omprison of α-oestrogen reeptor expression in ovrietomised nd intt femles reveled tht the higher perentge of neurons in whih IHC retion ws oserved ourred in ovrietomised femles. Oestrdiol dministrtion used in most of the exmined strutures, exept infundiulum nuleus nd ventrl prt of nuleus X, derese in the perentge of neurons in whih the retion ws oserved (2). In immunoretive neurons, the produt of immunohistohemil retion ws oserved in oth ell nuleus nd ytoplsm, ut the nulei of neurons were the min ple of retion. In our studies, expression of α- nd β-oestrogen reeptors ws demonstrted in ll regions of the mygdl. Detiled nlysis of the intrellulr distriution of retion mde it possile to isolte solterl nuleus s region of different sensitivity to endogenous level of oestrogen in rit mles, due to higher perentge of neurons in whih IHC retion for the presene of ER-β in ytoplsm ws oserved. Experimentl studies hve onfirmed tht lolistion of reeptor in the ell is vrile. Trnslotion of oestrogen reeptors from the ytoplsm to ell nuleus fter exposure to the hormone ws found (11, 17, 18). Dynmis of distriution of oestrogen reeptors were oserved fter tissue exposure to lignd whih, with referene to the otined results of our exmintions, indites tive physiologil proesses ourring in neurons of the mygdl with oestrogens prtiiption. Presene of reeptors in neurons n e interpreted in the wy tht IHC positive neurons re the trget of the hormone. 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