Testosterone 5alpha-reductase in discrete hypothalamic nuclear areas in the rat: Effect of castration

3396

347

TE,STOSTERONE~LPHA-REDUCTASEIN DhSCRETEHYPOTHALAMICNUCLEARAREA,SIN THE RAT: EFFECTOFCASTRATION Raberto C. Meicangl,FabloCeiotti,Paole NegrI-Cesl end Luclano Mortlnl Department of Enclo~inology, University of Mllmo, Via Andrm del ~-to 21, Milmo,ltaly Received 10-27-85

ABSTRACT The conversion of ted~-torone into 5alphe-dihydrotoatestorone (DHT) has been studied in different hypotI~lamlc nuclear areas en(I in the superficial levers of the cerebral cortex of normal and castratedmale rats.The tissue fragments utilizedin each incubation have been punched from frozen brain sectionsuUllzlno oallbratedneedles.Cestratlon has been performed 12 ( short term) end 180 (long term) days before ~ifice.The nuclear areas studied include:,the medial raoptio nucleus (MPN),the lateral preoptlc nucleus (LPN),the anterior hypothelemlc nucleus AHN), the lateral hypothelamic nucleus (LHN), the posterior hypothelamic nucleus (PHN),the nucleus ventremedlalIs (HYM),the ercuate nucleus (AR),the median eminence (ME),the nucleus perevantrieuloris (HPY), the suprmptlc nucleus (50) and the suprachlesmatic nucleus (SC). The poeslble effect of cestrltlon an the 5alphe-raductme,wera _e~_:-.3~_~_jin the MPN,LPN/~HN,LHN,PHN and in the cerebral cortex. The results indicatethet,in the male rat: 1) the lateral praoptlc(LPN) and the lateral hypothelemlc nuclel(LHN) Ix6~ a 5alphe-raauctlme activity higher than that present in the cerebral cortex end in the other hypothelamlc nuclei considered; 2)the suprachlaematlc nucleus (5C) apparently p,-~ a testosterone mat~llzlng activlb/lower than that found in any other nervous structures studied so far; 3) castration does not seem to Influencethe 5elphe-rt~luctaseactlvityeither in the hypothelamlc nuclear structures conside~ or In the cerebral cortex.

I

INTRODUCTION

The central nervous system (CK5) is presently consider~ to be a target structure for ~toe~one, since many important brain functions are modified or modulated by the exposure to the hormane,both in the pro- or pecinatal period and in adultL.~od( ! ).It is not totally deer so far, whether testosterone, in order to exert the full range of its ONS actions,should always be converted in the brain into its 5alphe-rad,__.~__metabolitos [ 17beta-bydro~/-Salphe-androstan3-one( DHT),5alphe-mdroatan- 3alpha, 17beto-dio1(3alpha-dio1)

and

5alphe-andrastan-

3beta, 17beto-dtol( 5beta-dtol)] or into estrogenic compounds (estredtol and estrane). However,it is notewort~ to underline that the ONSof ell mammalian and nonmammalian species studied so far,has been shown to be able both to 5alphe-raduce (1-4) end to aromatize androgens ( ! ,3 5); i.e.,to p . - ~

the two enzymatic systems whteh ore involved in the "activation" of testosterone in

the peripheral teroet structures (e.~,prostate,ssminel vesicles,Sertoli celts,etc.). These March, April 1985

Steroids

V o l u m e 46, N u m b e r s 3,4

~

348

"/' lI

]i,

0

X I)

l

enzymatic activitieshave been reported to be more concentrated in some brain areas (e.~ ,the hypothalamus,the llmblc system) than In others (6-8). In the peripheral er~-sensltIve

tissues and In the anterior pitultory,the formation of

DHT,the major 5alpha-reduced derivative of testoslere~,hes bean shown to be sensitive to ._er~cr__.Ins manipulations like castration and sex hormone administration (6,7,9,10). On the contrary,previous studies of this and other laboratories have shown that the 5alpha-reductese activity of the hypothalamus Is not modified by orchldectomy or by the administration of tmtmtorm~e and other sex steroids (7,11-14). Moreover,we heve previously demonstrated that a complete surgical'deafferentetlon" of the hypothalamus,or the administration of drugs (e.g., r__~'_,pine, parochloraphenylalonthe,atroplne,nalexone, morphine) known to interfere with the synthesis,the release or

the receptorlal

effects

of breln

neurotrensmitters

(like

catecholamines,sorotonln,the endogenousaplold paptides,atc.) do not affect the 5alphe-reducteso activity of the hypothelamus ( 15,16).

However,the r_nO~_Jbllitystill exists that this apparent

insensitivity of the hypothelamic enzyme to endocrine and ~ l n e

regulatory influences,

might be due to an ~tefact. It must be pointed out that the Influences of all these experimental manipulations on the 5alphe-reduct__e~e_ectlvty of the bypothelamus have been studied so far only In the tissue in tots ,andnot in the different morphologically and functionallywell-characterized nuclesr areas which constitutethis structure. It is then possiblethat the negative results obtained so far might be linked to some sort of'dilution" effect pro@__=~__by the presence,in the hylxAhelamus in tots,of a large pertion of metabolically inactive areas. In this connection,It is Interesting to recall that a report by Selmanoff et el.(17) has shown that,ln the rat,the lateral preaptic and the lateral hypothalarnlc nuclei,mlorodi-___,m~,ctm_amording to the punch technique of Palkovlte (18), = l b l t a higher 5alpha-_r_~r_i__,ctaseactivity than other hypothelamlc structures. The present series of experiments was designed : a) to exte~l the observations of Selmmoff at al. ( 17), by studying,in the normal adult male rat, the 5alphe-reduct___Aseactivity of a large group of mlcrodL__,~ected_hypothelamlc nuclei; and b) to verify whether castration might affect the 5alphe- _r_'e@__e_'-t__A~_ activity In some discrete hypothelamic regions. The nuclear areas studied

S

~

]

:iiO

X

:1) 1

349

Includa: the medial preoptic nucleus (MPN),the lateral preoptlc nucleus (LPN),the anterior

hypothelomlc nucleus (AHN), the lateral hypothelamlc nucleus (LHN), the posterior hypothelamlc nucleus (PHN),the nucleus ventromedlells (HYM),the ercuete nucleus (AR),the median eminence ( ME),the nucleus pereventriculeris (HPV), the suprasptlc nucleus (80) end the ouprechlasmetlc nucleus ( ~ ) . The effects of castration on the 5alpha-reduct_~__.were ~ , u p

to now,ln the

MPN,LPN,AHN,LHN,PHNand in the carebrel cortex. 1VL4~TER[ALS AND MJ~THODS Animals Male Sprague-Dawley rats (Charles River,Italy), weight 150-175 g, were used. The animals were mentalned In animal quarters wlth controlled tampereture end humldity.The light schedule was 14 h lightend 10 deck (lightson at 6:30 e.m.). Animals were fed e standard pellet dietend water was provided ed llbltum.Castretlon was performed under" lightether anesthesia 12 ( short term) and 180 ( long term) days before sau'ifice. All animals were killed by decapitation. MicrodL_~Ject__Ion of the hvoothelomic nuclei After" sacrifice,the brain was rapidly removed~ immediately frozen by Immersion in liquid nitrogen, then mounted on a cryostat and cut in 300 p serial frontal sections, through the hypothalamus.The tissues were kept at - 10"C threughout the whole procedure. Tissue fragments have been drawn under a stereomlcroscope , ~ i n g to the punch technique of Palkovits ( ! 8),utilizing calibrated needles with the Internal diameter of 0.3,0.6 and 1.3 mm, depending on the dimensions of the nuclei to be collected.In perticuler,1.3 mm needles were utilized for MPN end LPN,O.6 mm needles for AHN,LHN,PHN and the cerebral cortex,end 0.3 mm needles for' the remaining nuclei.The localizationend the identificationof each nucleus end the weluetion of their extension were performed e,z~=vding to the Ktlnlg and KIIppel (19) rat brain etlas;the initial section plane was A7190 I1. Incubation orecedures end detection qf"the matebolites Each incubation was performed on titus obtained from 2 rats for the nuclear areas drawn with the !.3 end 0.6 mm needles,endon tissue obtained from 4 rats for the others.The number of punches contained in esch incubation vial was in the ronge of ! 2 to 'tO,depenclIngon the extension of each nucleus. The cerebral cortex was drawn always from the first 3 s~ctions, 4 punches for each section. The lineorlty of the reaction between the samples containing the lowest end the highest amount of tissue was evaluated in preliminary experiments. Tissue punches were

Incubated In 250 pl of Krebs -Ringer buffer"solution in the pr----~e~-ceof a NADPH generating wstom(NADP, disodium salt, Boehringer Monnheim, 2mg;Olucc~e 6-Phosphete,disodlum salt,Boehrlngor Mennheim, I0 mg end OIc__zy~__6-Phesphete detp/dragonase from yesst grede 1,Boehringer Mmnheim,O.035 U.L); 14C t e s t o ~ , l O 4 dpm (,Specific activity-58 mCffmmol. ~kmershom England) wee ~._~.;.-:as the labelled substrata .The incubation was carried out at 37" C for 2 h in e Dubnoff metabolic shaker under a stream ofO2 / 002 98:2. At the end of the incubation,the reaction was stopped by ~ freezing the somplas.Tritlum labelled DHT (about 5000 dpm) was ~ to each sample in order to evaluate the recoveries;the metebolites formed were extracted with diethyl ethel- and separated on a thin layer aluminum oxide chromatography plate ( ( Merck 60 F254 type E,Darmstadt) end elutsd twice with e mixture of benzer~/absolute ethanol (97:3). The DHT spots were subsequently identifiedwith iodinevapoure, scraped off end the redi~tlvity was counted in e Packard 300 C liquid scintillation apectrometer.Quonch corrected ®m o1'the two isotopes were obtained by e calibration s t u d curve. DHT identity was

350

8

"Z' :IB :11, 0 I :1) I

proved In whole hypothelamus and cerebral cortex Incubates by recrystalllzetions to co~-~tant 3Hll4Cretio (Table 1).

TABLE I RECRYSTALLIZATION OF DHT TO CONSTANT SH/14C RATIO

3H/14(3 RATIO HYPOTHALAHU$ I ) Starting solution 2) First crystallization (absolute ethenel/wet~') 3 ) -_ _~¢or¢1_ _ crystallization

(acetone/water)

4) Third crystallization (diethyl ether/n-hexane)

0.22 0.23 0.23 0.23

CEREB~I. CORTEX

I) Startingsolution 2) Firstcrystallization(ebsoluteetI~nollwat~-) 3) ~:,:e
1.52 1.52 1.57 1.47

Protein content was evalueted,__~cox'_.ding to the method of Lowry et al.(20),in perallel samples,containing the same number of punches used in the 5alpha-reductase ~ . In the tables the date ere expr__~ed__as IXJ of DHT formed per ,ug of protein during 2 hours incubation period

Stgistics The date were enelyzed by one-w~/ermlysis of verience.To d~-mine the levels of signifioace of the responsos,the t-values were compared with the values of 8cheffe' and Dunnett tables for multiple comparison(21,22).

•Je ,,m ~ . o

x~

i

351

RESULTS Table 2 showsthe pg of DHT formed per I.[gof protein by eech nuclear hypotholemtcarm considered.

TABLE 2 DIHYDROTESTOSTERONE(DHT)FORI'IATIONIN THECEREBRALCORTEXAND IN DIFFERENT HYPOTHALAHICNUCLEARAREASOF THE BRAINOF NORMALHALE RATS Tissue

pg DHT/po protein

-Cerabral cortex

2.02*..0.30 (13)

.-MPN

2.22t0. !0 (10)

-LPN

3.62*_0.35 ( I 0)*

-AHN

!.87_.0.14 (7)

-LHN

3.89+_0.35 (8)*

-PHN

2.65+_0.41(4)

-HVH

2.88+_0.65 (5)

-AR

2.56_+0.16 (4)

-HE

2.30+0.53 (6)

-HPV

!.73+0.35(3)

-SO

!.56+0.33(2)

-SC

0.67+_.0.08 (2)*

*P
testosteroneinto DHT wi~ yields which are si~iflcont~ higher (P
than those found In the ~ W~lemtc

nuclei,the

nuclw ~ f o r m a t i o n of D ~

considered .in the I ~ o l

p~tlc

~

occurred also with yields s i ~ i f l ~ t ~

In the 1 ~ 1 hl~

than

352

~

-I- .m "It o i ~ r ~ I

those found In the cerebral cortex. It is interesting to note thet,wlth the exception of the lateral preoptlc( LPN),the lateral hypothelamlc(LHN) and the suprachlasmatic (SC) nuclei (see beio~ the 5alphe-reductase activity appeared to be quentitatlvely simller In all other hypothalemlc erees considered. The 5alphe-reductase activity of the supPachiesmatic nucleus(~) was found to be significantly (P
NORMAL pg/pg protein

CX~HORTTERM PO/Pg protein

CX LONOTERM pg/pg protein

Cerebral certex

2.02+0.30(13)

2.31+0.31 (12)

1.86+0.11(2).

-MPN

2.22*_0.10 ( IO)

2.27+0.26 ( I O)

1.72+0.12(2)

-LPN

3.62±0.35 ( tO)*

3.81 ±0.21 (9)*

2.40±0.49 (2)

-/din

1.87±0.14 (7)

1.93±0.21 (9)

2.14+0.6 (2)

-LHN

3.89+0.35 (8)*

4.58_,0.45 ( 10)*

2.56t0.07 (2)

-PHN

2.65_.0.41 (4)

2.57+_0.77 (4)

3.45± 1.23 (2)

*P
medial nuclei (P
that short term castration does not exert significant effects on the 5alphe-reducLe~e activlty of any of the hypothalamtc nuclei considered. The results obtained in long term castrated animals must be taken as being only indicative,since only two samples for each nucleer areas considered

8

~

|

!

IiO

353

I l I

muld ~ ana~r~ up ~ now. However It ~ p ~ r s t ~

also long =rm c~ratton ~es n~

st~lftcant~ Influence the ~ l p ~ - _r~_~t__~seactivl~ ~ the ~o~mlemlc nuclei c o n s l ~

DISCUSSION The results hero ___ec~r_lhed Indicate that,in the male rat : ! ) the lateral prsoptlc(LPN) and the

lateralhypothelamlc(LHN) nuclei possess____ a 5atphe-reductaseactivity higher then thatpresent In the cerebral cortex and In the other hypothelemlc nuclei conslder~I(MPN, AHN, PHN, HYM, AR, ME, HPV,SO) ; 2)the suprechlesmattc nucleus apparently ~

a testosterone metabolizing

~ l v l ~ lower t~n that found In any other nervous structures studied so far; and 3) c~tratlon does not seem to Influence the ~lp~-rmu~___~e_. activity either in the ~Ax~helamlc nuclear structures const~rm or In the cerebral cer~,x. The first ~ t ~ t~se resul~ ls In su~mtlal ~reement with a previous n~ort of ~ l m ~ f el.(17) whe ~

also ~own a hl~er ~lpm-rmuc~se ~ l v l ~

In the la~el p r ~ t l c

ia~'al ~ h e l e m i c nuclei t~n In the a t ~ ~x~helemic erms inclc_=~__In their = u ~ (m~ial pr~tic-m~lor

~ml~Ic

nuclei whl~

were d l ~ 3 ~

~ h e l e m u s , lncl~l~ the m~lan eminence;the w ~ the ventral mp~s ~ ~e ~ e m ~ i s l ~al,central,l~erel ~

nucleus ~

together~ :the mmimasal

nucleus;the ventrem~lal nucleus the emy~lotd complex, Incl~l~ the

mmlal emy~lold nuclei). It must he m ~ , ~ e v o r , t ~

the resul=

~re ___~s~_ 1 ~ ~ve ~ea ®~inm utllizl~ more p ~ e r ~ ~ I n m ~x~helemlc nuclei,~d usl~ ~ols ~ tissues m = l n ~ from on~ 2 or 4 mtmals. This result could ~ ~ l e ~ ~ ' e m e ssnsltlvl~ ~ the ~lphe- _~__~me m l ~ o ~ tel=lye lnsensltlvI~ ~ their ~ ,

t~nks = the

usm In the pr~e-~t =u~; _~$___~~ the

~lmm~f at el.(17) were f o ~

= use 19-25 r~s for

sl~le ~wmln~lon. At ~e memmt,lt Is =fftcult ~ un~rs~m ~e p~tol~lcal mmnl~ ~ the ht~er conom~lon ~ the 5alp~-rm~__~e__ ~ l v l ~ e - ~ n ~ r m In the I~eral p ~ t t c ~ x ~ l e m i c nuclei,since autoraciiosraphi¢'~tglJI~ ~ ~l~

t~n the m ~ l

a-~ In m n ~ t r = l ~

s[~vn t ~ t G " ~ brain ~ l ~

~

I~eral ere less

I ~ l l m ~s~s~rm~23,24). ~oreover,~

354

~

-~- 1,, ~ L o

•

Dim

rostral preoptlc area, the anterior hypothalamic nucleus and the medlobesal hypothalamus of the male rat ere ragordm es the crucial structures for the control of several neur(enmcrlna end beflavloral functions related to reproductive phenomena,lncludlng the feedback control of gonedotropin secretion(25). It might be opeculatsd that the lower uptake of labelled testmtertx~ In the lateral preoptlc and In the lateral hypothelamlc nuclei might be compensated by the Increased capacity of these structures to metalx)llze testosterone into Its more efficient metal]ollte DHT. However,it is also possible that the higher DHT conversion observed In the lateral preoptlc and hypothalamlc structures Is due to "contamination" of the lateral nuclei with the rnyellnated fibers of the medlal forebraln bundle which occupies large portions of the lateral hypothalamic end preoptlc areas. Indeed, our preYIous observations have six~wn that,ln the rat bratn,whlte matter structures {llke the corpus callosum and the optic tract which are mainly cam_~___ of myelinated flbers) are 3 to 5 times more active than the grey structures in 5alphe-raduclng testosterone(26). The pbys|ologlcal role of the formation of DHT In the white matter stucturas, which are largely devoid of androgen receptors,and which have not been considered up to now as posolble target structures for the action of ondrogens,romalns to be elucidated. The present experiments,performed on Isolated hypothelamlc nuclear structures, have confirmed the Insensitivity of the hypothelamlc 5alphe-reductese to the effect of deprivation ,shown by several previous authors utilizing the hypothelamus lrl toto( 7, i I - 14). Neither a short term nor a long term cestratlon was able to modify the 5alphe-reductaso actlvlty of the MPN,LPN/~HN,LHN and PHN. The prallminary results obtained In long term castrated animals Indicate that even 6 months of andrt~en deprivation do not effect the 5alpha-raductlon of testosterone In the structures considero(L The present data then confirm with a more sophisticated technique that the 5alpha- reduct__As?of the I'~/p~tlolBmus Is Insensitive to changes of the androgen env~ronment,,The present results are believed to provide a final answer to this ~__,,_~_Ion,since the experiments have been performed on Isolatedhypothalamlc nuclel In which the 5alpha-raducteso Is particularly ooncentrated~nd ~ t l y

eliminate the possibility of the'dilution'effect

Induced by the presence In the system of enzymatlcalty nonfunctional hypothalamic areas.

~T~=,~oz~i

355

ACKNOWLEDGMENTS The experiments here ___~_.tbed were supported by grants of the Constglto Nezlonale delle Ricerche,Roma,ltaly (through the Project "Preventive and Rehebtlitattve Medicine;' contracts no. 84-024:54-56-115-04980 and 84-02467-56-115-08178) end of the Mlniataro della Pubbllca Istruztone,Roma,ltaly.Soch support Is gratefully acknowL_adO~.___Thanksare due to Miss M.Ballabto and Dr. A.Polattl for their advice and esais fence.

REFERENCES 1. Martini, L., ENDOCRINEREV. 3.1 (1982).

2. CelotU, F., M__m~__,R.end Martini, L.,ln:Endocrlnoloav (De ~oot,L.d.,Edttor),Oruno and Stratton,New York( ! 979),pp 41-53. 3. Callard,O.V.,tn:Matabolismof Hormone1Stm'otds tn the Neuroe~tne Structures (Celottt ,F. ,Naftoltn,F. and Martini ,L.,Edltors),Raven Press,New York( 1984),pp 79102. 4. Lisboa,B.P., Franzen-Sievektng, M. and Breuar, H.,J. STER.BIOCHEM. 14,641 (1981). 5. Noftoitn, F., Ryan, K.d., Davies, I.d., Redo~/,Y.Y.R,, Flares, F., Patro,Z., Kuhn,M., White, R.J., Takaoaka,Y. and Woltn, L., REC.PRO0.HORM.RES.31,295 (1975). 6. M ~ , R., Stupnicka, E., Knlewald, Z. and Martini, L.,J. STER.BIOCHEM.3, 385 (1972). 7. Denef, C., Magnus, C. and McEwen, B.S., d. ENDOCRINOL.59,605 (1973). 8. Rommarts,F.F.C.and van der Holen, H.J., BIOCHIH.BIOPHYS.ACTA248,489 ( 197 ! ). 9. Celottl, F., Farraboe~i, P., Negrt-Cesi, P. and Hartinl,L., in: Hormonesand Ce,-=~-(dacobelll, $., Llndnor, N., Orlfflth, K., Editors), RavenPress, New York (1980), pp 431-442. ! O. Ce]otti, F.,Fartna,d.H.S. ,Santanlello,E. ,Martini ,Land Hotta,M, J.STER.BIOCHEH. I 1,221 (1979). 1 I. Kntewald, Z., Ham, R. and Martini, L., In: Hormonal Stm-olm (,James, V.D.H. Martini, L., Editors), Excerpta Medlca,Amsterdam (1971), pp 784-791. 12. Perez, A.E., OrUz, A, 0 ~ ,

M., Bwar, C. and Par~-Palac|oe, 0., STEROIDS25, 53

(1975). 13. Martini, L.,~lottl,F.,M~,R. and Motta,M.,d.STER.BIOCHEM.9, 411 (1978). 14. ~lottl, F.,Farlna,J.M.$.,Santanlello,E.,MartlnI,L.and Motta,M.,J.$TER.BIO(}IEM. LI.,215 (1979).

356

S

TE~ox~n'm

15. Celotti,F., Negri-Cest,P., Llmonta,P. end MelcmgI,C., d.$TER.BIOCHEH.I_..~.,229 ( t 983). 16. Celottt,F., Negrt-Cesl,P., Limonte,P., HolcmKjt,C.end Hartint,L., in: Hetah)11smof Hormone1$taroids In the NeuroendocrlneStructures (Celotti,F., Naftoltn,F. end Hartint,L., Editors), RavenPress, NewYork ( 1984),pp 35-52. 17. Selmenoff,M.K, Brodktn,L.D., Wetnar,R.I. end 5titert ,P.K, ENDOCRINOLOeY101,841 (1977) 18. Pelkovtts,H., BRAINRE~J~qCH53,449 (1973). 19. KOnto,d.F.R.and KIIppeI,RA., TI~ R~ Ornln: n 5tareotaxicAtlas of the Forebrntn end Lower Parts of the Brain Stem, The Williams andWllkins Compmy,Baltimore (1963). 20. Lowry,O.H., Rosebrough,N.d.,Farr~L. end Randall,R.d.,d.BIOL.CHEH. 193,265 (1951). 21. ,~x,.::_.~:(¢,O.W.endCochran,W.O.,3tattstlcal Hethods,The IowaState Unlvarslty Press, Ames (1967). 22. Dunnett,C.W.,JJq'I.STAT~.~. 50, 1096 ( 19551 23. 5ar,H. end Stumpf,W.E, in: Anatomical Neuroendocrinology(Stumpf, W.E. and

Lestar,D.O., Edltars), $.Kargar, Basel ( 19"75)_no -120--153. 24. Sharidm,P.d., CLINICALNEUROPI-IARI~. 7,281 (1984) 25. Slmpktns,d W., Kelra,5.P. and Kalra,P.8., ENDOGRINOLOOY112,665 ( ! 983). 26. Celottl,F., Maggl,R., Melcangi,C.R.,NegrI-CesI,P. and Martlnl,L., In: Endocrlnoloov (Lebrte,F. end Proulx,L., Editors), Elsevier/North Holland,Amsterdam (1984), pp 305-309.