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Neuroanatomical distribution of testosterone-metabolizing enzymes in the Japanese quail

Neuroanatomical distribution of testosterone-metabolizing enzymes in the Japanese quail
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  Brain Research, 422 (1987) 137-148 137 Elsevier BRE 12914 Neuroanatomical distribution of testosterone-metabolizing enzymes in the Japanese quail Michael Schumacher and Jacques althazart Laboratory of General and Comparative Biochemistry, University of Liege, Liege Belgium) (Accepted 3 March 1987) Key words: Testosterone; Aromatase; 5a-Reductase; 5fl-Reductase; Hypothalamus; Limbic system; Quail We describe a very sensitive and precise assay which allows one to study the metabolism of testosterone (T) in small brain nuclei dis- sected out according to the method of Palkovits and Brownstein. With this method, the neuroanatomical distributions of aromatase, and 5a- and 5fl-reductase activities were studied in adult male quail Coturnix coturnix japonica). The different enzymes show differ- ent neuroanatomical distributions. Production of estradiol-17fl (E2) was highest in the sexually dimorphic nucleus preopticus medialis (POM). We showed previously that the preoptic aromatase activity is higher in male than in female quail. As the POM is a central and very large structure within the preoptic area, the present results suggest a relationship between the neuroanatomical and the biochem- ical sex differences. By contrast, the production of 5a-DHT was highest in the lateral hypothalamic area (LHY), the bed nucleus of the pallial commissure (BPC) and the lateral septum (SL). The 5fl-reductase activity was highest in the lateral septum and in the ventral part of the archistriatum (AV). Moreover, there was a rostral to caudal decrease in 5fl-reductase activity in the hypothalamus. INTRODUCTION In male mammals and birds, testosterone (T) exerts some of its activating effects on behavior and gonadotrophin release through its metabolites 6'41'7°. In the brain, T is converted to active metabolites such as estradiol (E2) and 5a-dihydrotestosterone (5a- DHT) and to 5fl-reduced inactive compounds (5fl-di- hydrotestosterone = 5fl-DHT and 5fl-androstane- 3a,17fl-diol = 5fl,3a-diol; for review see ref. 5). Al- though aromatization of T to E 2 is critical for the acti- vation of male sexual behavior in quail 2 and rats s4, 5a-DHT also seems to play a role in this process in both species s,49. Treatment of castrated males with E 2 and/or 5a-DHT activates male sexual behavior (quail 79, rat 48) and inhibits the liberation of gonado- trophins (quail 2s, rat 68) whereas 5fl-DHT is inactive in this regard 28'4°. Thus 5fl-reduction of T is usually considered as an inactivation pathway for T 86. The synergism between E2 and 5a-DHT is not only due to an action of E 2 on the brain and of 5a-DHT on peripheral sexual characteristics and spinal motor neurons 17'52'78. Both hormones exert synergistic ac- tions at the brain level as shown by studies on the ac- tivation of copulation by intracerebral implants 12A8' 19 29. The control of vasopressin-containing fibers in the septum 3° by a synergistic action of E 2 and 5a- DHT also suggests that these steroids both act at the central level. By producing different amounts of active and inac- tive metabolites, the metabolism of T determines the sensitivity of the brain to the hormone. Sex differ- ences in brain sensitivity to the activating effects of T on behavior have been related to brain metabolism of T in quail 8°. Like female rats 61, female quail are less sensitive to the activating effects of T on behavior than males 1'7. As a biochemical correlate, T is also less efficient in inducing preoptic aromatase activity in females than in males 8°. The preoptic area (POA) and especially its medial part, is critically involved in Correspondence: J. Balthazart, Laboratoire de Biochimie G6n6rale et Compar6e, Universit6 de Liege, 17 place Delcour, B-4020 Li6ge, Belgium. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)  138 the neuroendocrine control of gonadotrophin release (rat 45, quail 26,27) and in the hormonal control of male reproductive behavior as shown with intracerebral hormone implants (rat 24'51, birds 9'10'34'39'73) and elec- trical stimulation (rat 54,58, pigeon3). The medial POA not only contains high levels of T- inducible aromatase (rat 75, dove 43'85, quail 8°) but also aigh levels of androgen and/or estrogen receptors in birds (quail 9°, other bird species, for review see ref. 4 and in mammals22'83). Moreover, many sexually di- morphic cell groups have been described within the POA of mammals 21'36'3s. A morphological sex differ- ence has recently been discovered in the quail POA 89. The nucleus preopticus medialis (POM), which is hormone sensitive (G.C. Panzica, C. Vig- lietti-Panzica, M. Calcagni, G.C. Anselmetti, M. Schumacher and J. Balthazart, unpublished data), is bigger in males than in females. Preliminary brain implant studies performed in our laboratory suggest that this nucleus is involved in the control of male sexual behavior. Thus the quail POA contains a sex- ually dimorphic enzyme (aromatase) and a sexually dimorphic nucleus (POM), which are probably both related to the activation of male reproductive behav- ior. The aim of the present study was to describe the neuroanatomical distribution of the T-metabolizing enzymes in the brain of the male quail and especially to see if the preoptic aromatase is located within the sexually dimorphic POM. For this purpose we used the brain microdissection technique described by Pal- kovits 66'67. We also studied the metabolism of T in other brain nuclei connected to the medial preoptic area in mammals and birds and involved in the con- trol of reproductive characteristics (for review in mammals see refs. 15, 48). Estrogens control female sexual behavior (rat 77, dove 35) and regulate the se- cretion of gonadotrophins (quail 2s, rat s6) by acting on the ventromediai hypothalamic nucleus (VMN). This nucleus sends inhibitory fibers to the POA whereas the POA sends inhibitory fibers to the VMN. As a consequence, lesions of the VMN facilitate male sex- ual behavior whereas lesions of the POA facilitate fe- male sexual behavior in rats 3t,32'71'74. This justified the study of VMN in the present experiments. Other nuclei studied were the bed nucleus of the pallial commissure (BPC), the lateral hypothalamic area (LHY) (both receiving fibers from the POM as has been shown in the pigeon15), the anterior hypotha- lamic nucleus (AM) and the nucleus periventricularis (PVN). The latter is characterized by dark-staining neurosecretory cells projecting to the median emi- nence (quailS9). Nuclei located in the avian equiva- lent of the mammalian amygdala (archistriatum ven- trale, AV; nucleus taenia, TN) and septum (medial and lateral parts) were also included in the study. The nucleus rotundus (RT) was taken as a control nu- cleus. The nomenclature of the different nuclei was that used by Kuenzel and Van Tienhoven 47. MATERIALS AND METHODS Experimental animals Animals used in the experiments were adult male quail (at least 7 weeks of age) obtained from a local breeder. They were individually caged under a 16 h light/8 h dark photoperiod (lights on at 06.00 h) for 2-4 weeks before sacrifice. Water and food were al- ways available ad libitum. The day before sacrifice, sexual behavior was tested with a receptive female for 5 min in conditions previously described 7. Only sexually active males were used for the present study. Fig. 1. Histological maps showing the localization and dissection of individual hypothalamic and limbic nuclei of the male quail. The thickness of successive sections was 200/~m. Black dots represent the internal diameter of punching needles (500, 800 and 1200/zm re- spectively). AC, nucleus accumbens; AM, nucleus anterior hypothalami; AV, archistriatum pars ventralis; BPC, bed nucleus pallial commissure; CA, anterior commissure; CO, optic chiasma; CPA, pallial commissure; DS, decussatio supraoptiea; E, ectostriatum; GLV, nucleus geniculatus lateralis; LA, nucleus laterali anterior hypothalami; LFB, lateral forebrain bundle; LHY, area lateralis hy- pothalami; NI, neostriatum intermedium; PA, paleostriatum augmentatum; PD, nucleus preopticus dorsolateralis; POM, nucleus preopticus medialis (a = anterior, b = posterior); PP, paleostriatum primitivum; PVN, nucleus paraventricularis; OF, quintofrontal tract; RT, nucleus rotundus; SL, nucleus septalis lateralis; SM, nucleus septalis medialis; IN, nucleus taenia; TSM, tractus septome- sencephalicus; V, ventricle; VMN, nucleus ventromedialis hypothalami. Nuclei were dissected out by a single medial punch (BPC, PD, POM, PVN, VMN) or bilaterally (AM, AV, LHY, RT, SL, SM, TN).  139 um um j V BPC o ~~ ~ .~.~::~ ~ es~~ii~ ~ 2 , , ~~~ r 400 .'.'.i...[": FB I~00~ 6 2 ~ 2200 '.': :.".: . L ~oo ~i i~ ._.-~.__..~ : ...~L-~ ~.T =-~. • 2.: .. B .. .~:. ,~, "" • ~ 4o~ ,ooo ~~~_ 1200 e SOOum 800um @ '---. ~ .~ i:;c 1200 um @ ~ Imm  140 icrodissection of the brain After decapitation, brains were quickly dissected out of the skull with small scissors and frozen on dry ice. Quick freezing of brains on dry ice does not af- fect the activity of T-metabolizing enzymes 8°. In Expt. 1 (validations), whole hypothalami were dis- sected out starting from the tractus septomesence- phalicus (TSM) to the level of the tuberal hypothala- mus (oculomotor nerves). For Expt. 2, specific brain nuclei were dissected out according to the method of Palkovits 66'67. The medulla-cerebellum area of the frozen brains was cut in the coronal plane with a steel razor thus provid- ing a fiat, well-oriented reference plane. The samples were mounted with Tissue-Tek (Lab-Tek Products, IL) on the specimen holder of a microtome with the forebrain up. The rostral end of the brain was then cut in a coronal plane with a steel razor approximate- ly 2 mm anterior to the TSM. Starting from this plane, coronal sections were made in a cryostat at -10 °C until TSM appeared completely with an in- verted V-shape. During this process, the plane of section was adjusted according to the stereotaxic at- las of the quail brain 13. The shape and symmetry of the ventricles, the ectostriatum, the paleostriatum primitivum, the tractus quintofrontalis as well as the beginning of the TSM were used to adjust the plane of section. Starting from the section where the entire TSM first appeared, brains were cut into thirteen 200 ktm serial sections (Fig. 1). Sections were thaw-mounted on glass slides and stored on dry ice until 4 brains had been cut. From these sections, individual nuclei were then isolated by punching them out with steel micro- dissection needles (inner diamters: 500,800 and 1200 um; see Fig. 1). In the first animals used to validate the dissection, the localization of the microdissec- tions was verified by staining the 200/~m sections with Toluidine blue 33. Individual nuclei from 3 to 4 birds had to be pooled for the assay of enzymatic activities (see Expt. 1). The punched area was blown out of the cold needle in a glass tube and pooled samples were homogenized in 100/A of ice-cold STMM buffer (0.25 M sucrose, 10 mM Tris-HCl, pH 7.4 at 20 °C, 5 mM MgCI2, 1 mM fl-mercaptoethanol) by ultrasonication (150 W, 15 s). Homogenates were immediately frozen in an acetone-dry ice bath (-78 °C). easure of enzymatic activites The radioenzymoassay for testosterone-metabo- lizing enzyme has already been described 42. For the assay, the 100/A homogenates were thawed in an ice- water bath. Immediately after thawing, 50 /A of STMM buffer containing [3H]testosterone (Radio- chemical Centre, Amersham, spec. act. 60 Ci/mmol, or New England Nuclear, spec. act. 141 Ci/mmol) and 3 mg/ml of NADPH 2 (final concentration: 1.2 mM = saturating levels sl) were added to the individ- ual tubes. Homogenates were then incubated at 40 °C for 15 min. Reactions were stopped by freezing samples at -20 °C. Steroids were extracted 3 times with diethyl ether after addition of 5/~g of carrier steroids and about 2000 counts of [4-14C]E2 (Amersham, spec. act. 50 mCi/mmol) and 2000 counts of 5a-[4-14C]DHT (Amersham, spec. act. 50 mCi/mmol) for calculation of recovery of estrogens and androgens respectively. Radioactive steroids were always repurified by thin layer chromatography (TLC) before use. After addition of 250/A of 1 M NaOH to the dry extracts, androgens were extracted 3 times with a mixture of toluene:cyclohexane (1:1, v/v). After neutralization of the aqueous phase by 1 M HCI, estrogens were extracted 3 times with diethyl ether. Both androgens and estrogens were separated by TLC on silica gel (estrogens: Macherey-Nagel No. 804023; androgens: Merck 5715) in a mixture of dichloromethane:ether (85:15, v/v). Androgens were revealed on the plates with Primulin (Sigma 7522) and estrogens with iodine vapours. The different metabolites were eluted in 100 ~1 of methanol and in 4 ml of Aqua-Luma and counted in a Tri-Carb 3255 Liquid Scintillation Spectrometer using the preset 3H/14C window. Counts were cor- rected for spillover, counting efficiency and tracer re- covery. The amounts of metabolites produced in con- trol tubes (blanks) containing buffer, NADPH 2 and tracers but no brain homogenate were subtracted from the final values. Four metabolites were quantified: estradiol-17fl (E2), 5a-dihydrotestosterone (5a-DHT), 5fl-di- hydrotestosterone (5fl-DHT) and 5fl-androstane- 3a,17fl-diol (5fl,3a-diol). The identity of these 4 me- tabolites has been confirmed for the quail hypothala- mus by recrystallizations to specific activity and/or constant isotopic ratio 42,sl. The amounts of metabolites formed by the differ-  mt brain nuclei were compared by ANOVA followed ~y Newman-Keuls tests when appropriate. ~ESULTS gxpt. 1: validations of the microdissection technique Precision and sensitivity of the assay. One aim of :he present experiment was to determine the preci- ;ion and the sensitivity of the radioenzymatic assay •hich would be obtained by using [3H]testosterone abelled in 6 positions as substrate ([1,2,6,7,16,17-3H IN)]T; New England Nuclear, spec. act. 141 Ci/ nmol) instead of the previously used [la,2a-3H]T ',Radiochemical Center Amersham; spec. act., 60 Ei/mmol; see refs. 80, 81). Whole hypothalami from 5 tdult males were pooled and homogenized by ultra- ;onication (150 W, 30 s) in ice-cold STMM buffer (fi- lal tissue concentration: 2 mg flesh weight (FW)/100 d STMM). One hundred #1 fractions of the homoge- late were then incubated in the presence of 50 MI of ~TMM containing NADPH 2 (final concentration: L.2 raM) and [1,2,6,7,16,17-3H]T (final concentra- :ion: 28 nM) for 5, 10, 15, 20 or 30 rain. The produc- :ions of E2, 5a-DHT, 5fl-DHT and 5fl,3a-diol were inear with respect to time for at least 15 rain. Values for the apparent K m and Vma x were then de- :ermined for the aromatase by incubating fractions of :he same homogenate for 15 min in the presence of tifferent amounts of testosterone with high specific ~ctivity (final substrate concentrations ranging from ) to 75 nM). The double reciprocal plot (Linewea- /er-Burke) of the E 2 production vs substrate con- :entration was linear in this range of substrate con- :entrations (r = 0.99). The kinetic parameters of the mzyme estimated from this plot (Vma x = 200 fmol/mg 'resh weight/h; apparent K m = 5.4 × 10 -s M) were in ~greement with previously published data for the ]uail brain 42's°'81. To compare the sensitivity and the precision of the tssay when using testosterone with low (60 Ci/mmol) md high (141 Ci/mmol) specific activity, the homog- mate was diluted to obtain different concentrations )f tissue (2, 1, 0.75, 0.50 and 0.25 mg fresh weight IFW)/100 #l of STMM buffer respectively). Fractions 100 #l) from the different dilutions were then incu- )ated at 40 °C for 15 rain in the presence of 50/A of ~TMM buffer containing one or the other radioactive estosterone both at the same molar concentration 141 TOTAL DPM 12000 _~ H 8000_ 4000_ L 0 0 05 1 2 ESTRADIOL 100_ 50 2 fmol/m9 FW/h O O O O 0 05 I 2 5a DIHYDROTESTOSTERONE I00 75 008o ° o BOO _ 50 4.000- L 2 0 i i i 0 05 1 2 0 ~S 1 53 DIHYDROTESTDSTERONE 120000_~ H B0000 _ 4.0000 _ L 0 , , , , 0 05 1 2 m9 FW St 0 05 o e Q • , t 1 2 mg FW Fig. 2. Formation of estradiol, 5a-dihydrotestosterone and 5fl- dihydrotestosterone observed during the in vitro incubation of different amounts of tissue (FW = fresh weight). Results are expressed as total DPM produced (left side) or as fmol/mg FW/h (right side). Each experimental point corresponds to the mean of triplicates. The dotted line at low tissue concentrations indicates undetectable production of metabolites. H = high specific activity testosterone (141 Ci/mmol = black circles) and L = low specific activity testosterone (60 Ci/mmol = open cir- cles). (final concentration: 28 nM) and NADPH2 (final concentration: 1.2 mM). Triplicate determinations were made in each condition. Ten identical 100/A fractions of the undiluted homogenate (2 mg FW/ml) were also incubated separately to determine the in- tra-assay variation of metabolite production with both types of radioactive substrate. Results of this experiment are shown in Fig. 2. The intra-assay coefficient of variation determined from the incubation of 10 fractions of the same ho- mogenate was about 10% for each metabolite and each type of substrate. The production of E2, 5a- DHT and 5fl-DHT were linear with respect to tissue concentration in the range from 0.25 to 2 mg. How- ever, the assay was at least twice as sensitive when
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