Molecular Brain Research, 14 (1992) 79-86 (~) 1992 Elsevier Science Pubhshers B.V. All rights reserved 0169-328X/92/$05 00

79

BRESM 70427

Molecular aspects of the regulation of tyrosine hydroxylase by testosterone M.E. Goldstein a'*, A.W. Tank b, L.H. Fossom b'** and R.W.

Hamill a

aNeurogerontology Umt, Departmentof Neurology, Monroe Community Hospital and bDepartment of Pharmacology, Untverstty of Rochester School of Medtcme and Denttstry, Rochester, NY 14620 (USA) (Accepted 21 January 1992)

Key words Tyrosine hydroxylase; Testosterone, Peripheral sympathetic nervous system, Gonadal steroid, Neurotransmttter enzyme, Autonomic nervous system

Previous studies have demonstrated that the sympathetic hypogastrlc gangha (HG) are dependent upon the continued presence of testosterone for normal development and maintenance of tyroslne hydroxylase (TH) activity. The regulation of TH by testosterone has been examined further to determine whether the reduction m TH activity following castration is associated wath changes in levels of TH protein and mRNA TH protein was measured by lmmunotltratlon of FIG homogenates using a TH-speclfiC antibody, and TH-speclfiC mRNA was detected by hybridization of dot blots of total RNA isolated from HG with a cDNA probe coding for TH. The results show that tyrosine hydroxylase activity, protein and mRNA are coordinately reduced in a graded fashion at 1, 2 and 4 weeks following castration Testosterone replacement therapy immediately following castration prevents the decrease m TH levels. The results indicate that gonadal steroids regulate the biosynthesis of T H m the HG Testosterone may control TH either directly by interacting with neurons of the HG, or indirectly by altering levels of trophic factors in the target tissues. INTRODUCTION Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines in the sympathetic nervous system, is regulated by a n u m b e r of factors. In the peripheral nervous system, transsynaptic neurotransmitter release alters T H activity and protein in postsynaptic neurons and these effects are o b s e r v e d with electrical stimulation of preganglionic nerves, t r e a t m e n t with reserpine, or exposure to cold stress (for reviews, see refs. 4, 60, 61). F u r t h e r m o r e , the increase in enzyme protein and activity appears to be associated with an increase in m R N A encoding T H 5'15'49. In general, T H appears to r e s p o n d by either a rapid, short-term activation of pre-existing enzyme molecules or a delayed long-term increase in the synthesis of new enzyme molecules (reviewed in refs. 60, 61). O u r particular focus has been the role of h o r m o n e s in regulating T H activity in p e r i p h e r a l sympathetic ganglia. The induction of T H by glucocorticoids and c A M P has been well characterized. Glucocorticoids and c A M P induce T H m n e u r o b l a s t o m a 51'54 and p h e o c h r o m o c y t o m a cells2"29. Tyrosine hydroxylase protein levels, the rate of

synthesis of T H protein 48 and levels of T H m R N A 3°'49 are altered in p h e o c h r o m o c y t o m a cells as a result of increased transcription of the T H gene 17'29. In the central nervous system, a corticosterone-induced increase in T H activity has been observed in the mouse locus coeruleus 34. Tyrosine hydroxylase in peripheral and central autonomic pathways may also be regulated by such gonadal steroids as androgens 14'15'22'24, estrogen, and progesterone 2°'53. Castration of male rodents results in reduced levels of tyrosine hydroxylase activity in the superior cervical ganglion 24"38, hypogastric ganglion 22 and arcuate nucleus 9. These results suggest that androgens maintain elevated levels of T H in both peripheral and central neurons. In contrast, the effects of estrogen on T H activity are complex. Treatment of ovarectomized female rats with estrogen and p r o g e s t e r o n e results in increased levels of T H activity in the m e d i a n eminence 2°' 53 whereas estrogen t r e a t m e n t of ovarectomized rats resuits in reduced levels of T H activity in the b a s o m e d i a l hypothalamus 32, arcuate and periventrlcular nuclei 41 and superior cervical ganglion 1°. In addition, T H m R N A and gene transcription in the arcuate nucleus are decreased following acute t r e a t m e n t with estrogen s .

* Present address Laboratory of Neurochemistry, Building 36, Room 4D22, NINDS, NIH Bethesda, MD 20892, USA ** Present address: Department of Biochemistry and Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA Correspondence R W Hamlll, Neurogerontology Unit, Neurology Department, Monroe Commumty Hospltal/Umverslty of Rochester School of Medicine and DenUstry, 435 East Henrietta Road, Rochester, NY 14620, USA

80

TH

regulanon

by gonadal

s t u d i e d in t h e h y p o g a s m c

stermds

ganghon

has recently been

( H G ) zz 36 37 zo. C a s -

t r a t m n o f a d u l t m a l e r a t s r e d u c e s T H a c t i v i t y in t h e H G suggesting that testosterone non of TH m penperal inherent which TH

m

p l a y s a r o l e in t h e r e g u l a -

sympathetic

all o f t h e s e

studies

a c t i v i t y ~s a l t e r e d

further explore the mechanism dress whether

The

ganglia. A question

is t h e

mechamsm

present

by

experiments

of this regulation and ad-

t h e d e c r e a s e xn T H a c t i v i t y f o l l o w i n g c a s -

tration represents

reduced

biosynthesis of enzyme

ecules and altered levels of mRNA

mol-

for the enzyme

MATERIALS AND METHODS

E~pertmental atllmal~ Adult, Sprague-Dawley male rats (59-65 days old, 250-300 g) ~ere obtained from Charles Rwer Labs They were housed 2-3 rats per cage, allowed free access to food and water, and were exposed to 12 h of light daffy in a temperature-controlled environment (22 _+ I°C) Rats were killed by exposure to CO 2 fumes ~mmedmtely prior to assay

Surgical treatment Castranon Rats were anesthetized with 3 0% halothane In 100% oxygen and recto,rod rather bilateral castranon or sham operation For sham operanon, the scrotal sac was opened, exposing the testis, epldldymls and vas deferens, and closed with metal clips Castration preserved the eDdldymls and ,~as deferens Ganglia dissection The hypogastnc gangha are comprised ot 2 4 ganghon whmh innervate the ,~as deferens, seminal vesicles, ejaculatory duct and prostate They are located at the a r b o n z a n o n ol the deferential artery, where the '~as deferens and seminal vesicle join to form the elaculatory duct They were removed with the aid of a dissecting m~croscope Drug treatment For testosterone replacement studies, rats were rejected on the day of castraUon and every week until sacrifice with rather 0 1 ml ~ehmle (soy bean oil) or 20 mg/kg testosterone enanthate in 0 1 ml vehicle Biochemical procedto es

T~tostne hydroD,lase activity Tyrosme hydroxylase actwlty was assayed as described previously 4° with modlficatzons Trmated tyroslne-labeled side chain was obtained from A m e r s h a m , Arlington Hmghts, IL A 10 !d aliquot of nssue h o m o g e m z e d m water at a rano of 1 ganglia to 50-100 id was added to 15 Ftl of a solunon containing in final concentration 4 4 itM [3H]tyrosme (spec act 9 0~ Ct/mmol), 100 m M 2-mercaptoethanol, 2 0 m M tetrahydro-tbloptenn, (I 15 m M NSD 1055 (Sandev), 2 5 m M ferrous sulfate and 0 32 M potassium phosphate buffer at a final pH of 6 0 The reaction was terminated after 8 m m at 37°C by the a d d m o n of 2511 ul of 0 4 M perchlonc acid containing 21~g/ml e-dlhydroxyphenylalanme (L-DOPA) and d o p a m m e To this mixture was added 5 0 ml of a solution at a final pH of 8 6 - 8 8 containing 0 3 g alumina m 0 04 M Tns, p H 8 6. 0 04 M s o d m m E D T A , 0 06 M N a O H and {1 07 M potassium phosphate, pH 8 6 After two successive washes with 5 ml of 0.005 M Tns, p H 8 6, the L-[~H]DOPA was eluted with 1 5 m l o f 1 0 M aceticac~d The eluate was counted in a h q md scintillation counter m 10 ml of Scmtwerse I Fvrosme hydro.tylase protein Tyrosme hydroxylase protein was measured by lmmunontratlon as described previously 6 49 with mod~tmatlons Pa~rs of ganglia were homogenized in 150-200 itl water ,uld 21) ld ahquots were incubated with 10 m M Tns, pH 7 5 and 11 t)% saline or serial dilutions (1/10-1/80) of a polyclonal antibody, produced in a rabbit against bovine tyrosme hydroxylase, diluted in 10 m M Tns, pH 7 5 and 0 9% saline such that the final volume was 30 !tl The mixtures were incubated for 1 h at room tempera-

ture with m t e r m m e n t agitation Twenty-five ~1 of a 10% suspension ol Staph aureu~ cells (Enzyme Center) was added to each mixture and incubated 15 m m at 4°C The mixtures were centrifuged m an Eppendorf mlcrocentnfuge at 10,000 × g for 5 m m and duphcate 10/A ahquots of the supernatant were assayed for T H activity as described above In prehmlnary experiments, normal sheep serum used as a n e g a m e control In the place of the TH-spec~fic polyclonal antibody did not prempltate any T H protein Total protein was measured b~ the method of Lowry et al ~* l~olatton o [ m R N A Tyrosme hydroxylase m R N A was ~solated by the method ot ChomczynsM and Sacchfl t Hypogastrlc gangha were removed from 5-10 rats at each time point examined, pooled and homogenized in 500 /d of 4 M g u a m d m e lsothlocyanate (Boehrmger M a n n h m m Blochemmals), 25 m M s o d m m citrate, pH 7 0, 0 5% sarcosyl and 100 m M 2-mercaptoethanol Fifty itl 2 M s o d m m acetate, pH 4 0, 500 l,l phenol and 2 0 0 / d chloroform lsoamyl alcohol (49 1) were added to the homogenate, mixed by inversion after each a d d m o n , shaken vigorously and left overmght at -20°C The mixture was centrifuged in an Eppendorf mlcrocentrlfuge at 15.000 × g tor 30 ram, the upper aqueous layer containing the R N A was r e m m e d and 1 ~olume of me cold lsopropanol was added, mixed well and left at -20°C for 1 h The tube was spun m a mlcrocentnfuge at 15,000 × g for 30 m m and the pellet was reh o m o g e m z e d in l) 3 ml of the same 4 M guamdlne ,sothmcyanate solunon to which 1 volume of tsopropanol was added. This mixture was left at -20°C for 1 h and the pellet was resuspended m 500 gtl DEPC-treated water and q u a n n t a t e d for m R N A at 260 nm The R N A was stored at -80°C in ahquots Quanntatton o f m R N A Tyrosme hydroxylase-specafic m R N A was quanntated by dot blot analysis Total m R N A was incubated for 15 m m m 20× SSC (0 3 M s o d m m citrate, 3 M s o d m m chloride) and formaldehyde at a ratio of 3 2 at 65°C. and serial dllunons were apphed to nitrocellulose beginning with l ftg in the first dot The blots were baked at 80°C for 2 h, prehybrldlzed in 50% formamlde, II 1% SDS, 5× Denhardt's solution, 4× SSPE, and 0 1 mg/ml salmon sperm D N A and hybridized in fresh h y b n d l z a n o n buffer with 0 25-0 5(1 × 10~' cpm/ml ot a KpnIlPstI 300 bp tragment of p T H 4 c D N A that encodes the 3' end of T H m R N A 3° The blots were washed at room temperature with two changes of 2× SSC, 10 m m each, then with two changes each o[ 1× , 0 5× , 0 2 and 0 1× SSC, for 10 m m each wash, all at 50°C The blots were exposed to Kodak X - A R film at -80°C for 2 - 4 days with intensifying screens and autoradlograms were q u a n n t a t e d by densltometry In order to compare autoradmgraphm data, the densltometer was zeroed on the background of the respective film and percent change m m R N A levels was d e m e d from the linear portion of the curves Stattvtws Data were analyzed using Student's t-test and expressed as mean _+ S E M Mulnple data were analyzed with the one-way analysis of variance and the Flsher's PLSD test RESULTS The HG of adult male rats were removed for

TH

RNA

enzyme

(mRNA),

activity,

TH

protein,

and assayed

TH

messenger

a n d t o t a l g a n g l i o n p r o t e i n at 1 w e e k , 2

weeks and 4 weeks following bilateral castration.

Tvrosme hydroxylase acttvtt~' Tyrosme hydroxylase tration

decreased

tiwty measured

on separate

factor of two, m order assays performed by normalizing

activity m the HG following cas-

slgmficantly over time. to compare

on separate

Since TH

ac-

days may vary by up to a data obtained

from

days, results were pooled

the activity of castrates

expressed

as a

percent of the respective controls. TH total activity was

81 06TH actwity in the hypogastric ganglion

a. 150

O

co

05"

e-

04'

a.

1 WEEK

CGNIROL

03"

100

¢-

c O o O

o~

o

50

E

01

e-

00

02

00 1 WEEK

2 WEEKS

04

06

08

10

4 WEEKS

06 c O

E~

b.

TH specific activity In the hypogastrlc ganglion

t-

J~

~ .

~

04"

o~

15o

2 WEEKS

05"

03"

J-

~

~

.

•

CASTRATE

02" o

100

E

01

t-

00 00

02

04

06

08

1

50

1 WEEK

2 WEEKS

05

e"~

0 4 L I 03

4 WEEKS

Fig. 1. Tyrosme hydroxylase (TH) actwlty m the h y p o g a s t n c ganghon following castration of adult male rats a total ganghon T H activity (nmol/h/ganghon, m e a n + S E . M . ) is reduced to 64.9 + 11 9% (P < 0.025, n = 7), 30 8 + 6.1% (P < 0 05, n = 4) and 21 8 + 2 9% (P < 0.005, n = 8 for controls, n = 7 for castrates) of controls at 1, 2 and 4 weeks following castration, respectwely b T H specific activity (nmol/h/mg protein; m e a n + S.E M ) is reduced to 89 2 + 20 7% (not slgmficant, n = 7), 32 7 + 9.6% (P < 0.05, n = 4) and 44 6 + 6.4% (P < 0.005, n = 8 for controls, n = 7 for castrates) of controls at 1, 2 and 4 weeks following castration, respectively. Controls, sohd bars; castrates, slashed bars Average values for control total g a n g h o n T H activity at each t~me point were 0 269, 0 542, and 0.321 nmol/h/ganglion, respectively.

reduced to 64.9 + 11.9 (P < 0.025), 30.8 + 6.1 (P < 0.05) and 21.8 + 2.9 (P < 0.005) percent of control values 1, 2 and 4 weeks following castration, respectively (Fig. la). T H enzyme-specific activity, measured to compare the change in T H activity with total ganglion protein, was reduced to 89.4 + 20.7% (not significant), 32.7 + 9.6% (P < 0.05) and 44.6 + 6.4% (P < 0.005) of control values 1, 2 and 4 weeks following castration, respectively (Fig. lb).

Tyrostne hydroxylase protein Tyrosine hydroxylase protein levels were measured by immunotitratlon. T H protein was precipitated from homogenates of H G with increasing concentrations of a TH-specific polyclonal antibody, and enzyme activity remaining in the supernatants was measured and expressed

t

to

m

4 WEEKS

C.

-~o--•

J~

o

E

coNn:~x.

CASTRATE

ol

c"

O0 00

02

04

06

08

10

ul antibody Fig. 2. Tyrosme hydroxylase (TH) protein m the hypogastnc ganghon following castration of adult male rats I m m u n o m r a t l o n curves of T H total actlwty expressed as nmol/h/ganghon; m e a n + S.E M. remaining m the supernatant w~th increasing a m o u n t s of TH-speclfic antibody at 1 week (n = 4) (a), 2 weeks (n = 4) (b), and 4 weeks (n = 3) (c), following castration Controls, open squares; castrates, sohd squares

as total ganglion activity (Fig. 2a-c). Since data obtained from assays performed on separate days showed nearly identical results, representative titration curves are shown. For each immunotitration, the enzyme activity remaining in the supernatants decreased with increasing concentrations of antibody. At 1, 2 and 4 weeks following castration, the curve was shifted downward and to the left, indicating that there was a reduction in the amount of T H enzyme protein. The amount of antibody required to reduce T H total activity in the supernatant by 50% (ABs0) was measured for each homogenate and the ABs0 from castrates was expressed as a percent of the ABso from controls. The ABso values (Table I) de-

82 TABLE I

and 4 weeks following castration (Cx), respectively.

4Bs0 i'alue~ (the amount of antibody required to reduce TH acretry Ol the ~upernatant by 50%) extrapolated from the trnmunotttratlon ctttl'e; off TH total actn'ttv following castration

H o r m o n a l restoration o f T H actn'tty, p r o t e m a n d m R N A

% of conttol

ABe,,

In o r d e r to test w h e t h e r t h e e f f e c t s o f c a s t r a t i o n o n the T H indices w e r e s e c o n d a r y to t e s t o s t e r o n e d e p l e t i o n , a n d r o g e n r e p l a c e m e n t t h e r a p y with t e s t o s t e r o n e e n a n -

1 week 2 weeks 4 weeks

Control

('a~trate

(J 176 0 359 0 216

I) 114 0 124 0 065

t h a t e ( T E l was t m t i a t e d

Rats were castrated and imme-

diately r e c e i v e d e i t h e r 20 m g / k g t e s t o s t e r o n e e n a n t h a t e

64 8 34 5 30 I

a.

TH activity m the hypogastric

c r e a s e d to 64 8, 34.5 a n d 30.1c~ oI c o n t r o l s at 1 w e e k , 2

ganglion

250

weeks and 4 weeks, respectively Therefore, T H protein a n d actlvlty d e c r e a s e d m parallel f o l l o w i n g c a s t r a t i o n

200 > U

Tvrostne hydroxylase m R N A

150

Total m R N A was i s o l a t e d f r o m p o o l e d H G o f c o n t r o l

0

a n d c a s t r a t e d rats at 1, 2 a n d 4 w e e k s f o l l o w i n g castra-

tO U

100

tion. T h e a m o u n t o f R N A e x t r a c t e d f r o m ganglia f r o m 50

c o n t r o l a n d e x p e r i m e n t a l a n i m a l s did n o t significantly differ D o t b l o t s o f serially d i l u t e d m R N A w e r e h y b r i d -

0

ized w~th a c D N A p r o b e e n c o d i n g t h e 3' r e g i o n o f T H m R N A 3" (Fig. 3)

CONmO_

This p r o b e h y b r i d i z e d to a single

CASTRATE

CASTRATE CASTRATE

m R N A b a n d o f 1,800 to 1,900 b p c o r r e s p o n d i n g to the mRNA

c o d i n g for T H o n N o r t h e r n b l o t s o f poly A +

mRNA

i s o l a t e d f r o m H G ( n o t s h o w n ) . F o l l o w i n g cas-

t r a t i o n , T H m R N A in t h e H G w a s r e d u c e d as early as 1

b.

+

+

]E

OIL

TH specific activity in the hypogastric

ganglion

w e e k a n d r e m a i n e d at r e d u c e d levels t h r o u g h 4 w e e k s A r e p r e s e n t a t i v e d o t b l o t is s h o w n (Fig. 3) D e n s a t o m e t -

150

tic analysis o f t h e s e d o t b l o t s , utilizing t h e h n e a r p o r t t o n

>

of the curve, showed that TH-specific m R N A

U

was re-

d u c e d to 61.9, 51.1 a n d 18.5% o f c o n t r o l values (C) 1, 2 °~ U

100

@ Q.

1Week

2Weeks

4Weeks 0

C

Cx

C

Cx

C

Cx

L. c 0 u

50

CASTRATE

Fig 3 Tyrosme hydroxylase (TH) mRNA in the hypogastrlc ganghon following castration of adult male rats Serial dilutions of total RNA isolated from 5-10 pooled gangha at each time point on mtrocellulose filters hybridized with a cDNA probe specific for TH mRNA TH mRNA is reduced to 61 2, 51 1 and 18 5% of controls (C) at 1, 2 and 4 weeks following castration (Cx)

~TE

~TE

+

+

TE

OIL

Fig 4 Tyroslne hydroxylase (TH) activity in the hypogastrlc ganghon at tour weeks following castration and treatment with testosterone enanthate a TH total activity (nmol/h/gangllon, mean _+ S E M ) is reduced to 3l 8c~ of controls (P = 0 001, n = 7) in castrated rats with no treatment and to 34 0c~ of controls (P < 0 005, n = 9) m castrated rats treated with vehicle In rats treated with testosterone, TH acnvlty increases to 185 4% of controls (P < 0 025, n = 8) b TH-speclfiC acuvny (nmol/h/mg protein, mean + S E M ) is reduced to 55 2% of controls (P < 0 01, n = 7) in castrated rats with no treatment and to 62 5% of controls (P < 0 05, n = 9) m castrated rats treated with vehicle In rats treated w~th testosterone, TH-speclfiC actl~ny Ls 116 0% of controls (no significant change, n = 8) The average value for control TH total activity was 0 196 nmol/h/ganghon

83

TH protein

in the hypogastric

ganglion

CASTRATE CONTROL

CASTRATE

CASTRATE

+

+

TE

OIL

o6

.9~-

05

e-

04

~

o3

o

02

c-

~T

• •

~

CASTRATE CASTRATE+TE

o

E

01

o

oo

, 00

02

04

06

.

, -.-7--08

10

~tl antibody Fig 5 Tyroslne hydroxylase (TH) protein m the hypogastric ganghon at 4 weeks following castration and treatment with testosterone enanthate Immunotltratlon curves of T H total actw~ty expressed as nmol/h/ganghon remaining m the supernatant with increasing a m o u n t s of TH-speclfic antibody at 4 weeks following castration Controls, open squares (n = 10), castrates, sohd circles (n = 6), c a s t r a t e s + o d , open orcles (n = 8), c a s t r a t e s + T E , sohd squares (n = 8)

in soy oil as vehicle ( c a s t r a t e + T E ) , vehicle alone ( c a s t r a t e + o i l ) , or no t r e a t m e n t (castrate). F o u r weeks following castration the hypogastric ganglia were removed and assayed for enzyme activity, protein and m R N A . Total ganglion T H activity was significantly reduced to 31.8% of controls ( P = 0.001) in castrated rats with no t r e a t m e n t (castrate) and to 34.0% of controls ( P < 0.005) in castrated rats treated with vehicle alone (castrate+oil). In rats treated with testosterone following castration ( c a s t r a t e + T E ) , T H activity was not only restored, but actually increased to 185.4% of controls ( P < 0.025) (Fig. 4a). T H specific activity was r e d u c e d to 55.2% of controls ( P < 0.01) in castrated rats with no t r e a t m e n t (castrate) and to 62.6% of controls ( P < 0.05) in castrated rats t r e a t e d with vehicle alone (castrate+oil).

T A B L E II

ABso values (the amount of antibody required to reduce TH activity m the supernatant by 50%) extrapolated from the tmmunotttratton curves of TH total activity following castration and testosterone treatment

Control Castrate Castrate + T E Castrate + od

ABso

% of control

0 165 0.070 0 340 0 070

100 0 42 4 206 1 42.4

Fig. 6 Tyrosme hydroxylase (TH) mRNA m the hypogastric ganghon following castration and testosterone treatment of adult male rats Serial ddutlons of total RNA on nitrocellulose filters hybridized with a cDNA probe specific for TH TH mRNA is reduced to 23.9% of controls m castrates with no treatment (Cx) and 32 4% m castrates treated with vehicle (Cx+od), and is partially restored to 46 5% of controls In castrates treated with testosterone (Cx+TE).

The increase in specific activity in rats treated with testosterone ( c a s t r a t e + T E ) following castration is 116.0% of controls (not significant) (Fig. 4b) indicating that the portion of the testosterone-induced increase in total ganglion T H activity above 100.0% (Fig. 4a) is associated with an overall increase in total protein. Tyrosine hydroxylase protein levels in the H G were m e a s u r e d by immunotitration and expressed as total ganglion T H activity remaining in the supernatant (Fig. 5). The curves from rats with no t r e a t m e n t (castrate) and from rats treated with vehicle alone ( c a s t r a t e + o i l ) are shifted downward and to the left as c o m p a r e d with the control curve (control). The curves from rats treated with testosterone, however, are shifted upward and to the right indicating that T H protein is increased well over control values. The ABs0 values (Table II) demonstrate that T H protein decreases to 42.4% of control levels in both the no t r e a t m e n t (castrate) and vehicle treatment ( c a s t r a t e + o i l ) groups but increases to 206.1% of controls when treated with testosterone ( c a s t r a t e + T E ) . Total m R N A was isolated from the H G of control rats and rats which were castrated and treated with testosterone, with vehicle alone, or with no treatment. The R N A

84 was serially diluted on nitrocellulose and hybridized with the TH-specific cDNA probe (Fig. 6). Densitometric analysis of the blots indicated that TH mRNA was reduced to 32.4 and 23.9% of controls in castrates treated with vehicle (Cx+oil) or with no treatment (Cx), respectively. In HG of rats which were castrated and treated with testosterone (Cx+TE), TH mRNA levels were partially restored to 46.5% of controls. These experiments, utilizing pooled ganglia from 5-10 rats in each group, were repeated three times. Each experiment revealed similar results. DISCUSSION Tyrosine hydroxylase enzyme activity, protein and mRNA in hypogastric ganglia of the adult male rat sympathetic nervous system are coordinately reduced in response to reduced levels of circulating androgens following castration. These results confirm and expand previous findings of reduced TH activity in HG following castration of developing36'37"4° and adult 22 male rats. The present experiments have further established that the decrease in TH activity following castration represents reduced biosynthesis of enzyme molecules. Despite the immediate fall in plasma testosterone levels, which occurs within 24 h TM, the decreases in TH mRNA, protein and activity are gradual, reaching stable levels only between 2 and 4 weeks following castration. The 30 h half-life of TH protein 49 probably contributes to the gradual decline and allows for some of the activity to linger. In the hypogastrlc ganglion, TH specific activity, as well as total ganglion activity, decreases following castration and is restored to control levels with replacement therapy. The effect of castration, therefore, is specific to TH as opposed to only a generalized effect on total ganglion protein. However, since increases above control levels observed in TH activity following replacement therapy no longer exist when expressed as specific activity, testosterone replacement does produce an effect on total ganglion protein, as well as an effect specifically on TH gene expression. These observations in adult animals utilizing a hormonal paradigm mirror similar data from earher developmental stu&es. During ontogenesls, the rise in TH activity in the superior cervical ganglion of male rodents is the result of an increase in TH specific activity7 and this increase in TH activity has been shown to be the direct result of an increase in the number of TH enzyme molecules ~. Similarly, studies of TH induction in adult rodents indicate that increases in TH activity are associated with increased number of enzyme molecules 28. Reduced TH protein and activity levels following castration are the result of reduced TH mRNA levels, due

to either a decreased rate of transcription of the TH gene or altered stabihzatlon of TH mRNA TH gene transcription, as measured by nuclear run-on assays, is increased following treatment of pheochromocytoma cells with glucocorticoids and cAMP 17'29 These alterations in TH gene transcription are reflected in changes in levels of TH mRNA, protein and activity 2'3°'48'49 Steroid hormones also affect mRNA stabilization ~245; this effect might result in a change in levels of TH mRNA, protein and activity in the absence of any change in the level of transcription. Despite the complete restoration of TH protein and activity with testosterone replacement therapy, failure of TH mRNA to be completely restored may indicate that the observed increase of TH mRNA following testosterone treatment is suffioent to restore TH protein and activity. Induction of TH protein and activity is not always reflected in equivalent changes in levels of TH mRNA 46'49. Other posttranscrIptional mechanisms which regulate the level of translation of TH mRNA may result in increased levels of TH protein in the absence of any change in the level of TH mRNA. The aforementioned neurochemical observations support previous studies which indicate that gonadal steroids have widespread effects on neurons of the peripheral sympathetic nervous system. Compared to females, adult male rats have greater numbers of neurons in the 5CG57 5~ and major pelvic ganglion (PG) 21. Both testosterone and estrogen treatment of neonatal male rats results in increased neuron and synapse numbers In the SCG 57'58, the testosterone effect may be via the aromatization of testosterone to estradiol ss 56. Recently, the significance of sexual dimorphlsm in the SCG has been questioned since neuron number in the ganglia may vary substantially within genders and male-female differences may not differ significantly ~6 However, methodological issues preclude exact comparisons of data and the authors did note a trend of increased neuron numbers in the SCG of male animals 16 Normal development of neuron size and catecholamine content in the (PG) is inhibited by castration 44 and Increased by postnatal treatment of male rats with testosterone 25'43 In addition, testosterone propionate and deithylstilbestrol treatment increases the number of neurons in the PG 47. In the hypogastric ganghon, castration of male rats at 10-11 days of age results in a reduction in neuron size with no change in neuron number 3~ whereas castration at birth reduces neuron number by about 60e/c-~5 Changes m levels of TH synthesis and activity may result from the direct interaction of androgens with the hypogastric ganghon and subsequently the TH gene Hypogastric ganglia contain cytosolic androgen receptors which bind androgens with saturable, specific, and high affinity binding 35, indicating that the interaction of an-

85 d r o g e n s with t h e H G m a y in fact be a direct o n e . Ty-

tissue of the S C G , and a l t e r e d levels of N G F in the sub-

rosine h y d r o x y l a s e in c a t e c h o l a m i n e r g i c n e u r o n s of the S C G is r e s p o n s i v e to e s t r o g e n 1° and a n d r o g e n s 13'14"24"3s,

m a x i l l a r y gland m a y indirectly influence the biosynthesis of T H in the S C G 7'24. T h e effects o f t e s t o s t e r o n e o n T H

and p o s t n a t a l c a s t r a t i o n of m a l e rats p r e v e n t s the nor-

in S C G and o n s u b m a x i l l a r y gland w e i g h t parallel o n e

mal d e v e l o p m e n t a l i n c r e a s e in T H activity in the m a j o r pelvic g a n g l i o n ( P G ) 39. It is possible, t h e r e f o r e , that T H

a n o t h e r , again suggesting an indirect r e s p o n s e of T H to t e s t o s t e r o n e via N G F 13'14'38. A n d r o g e n r e c e p t o r s exist

in a n u m b e r of a u t o n o m i c ganglia m a y r e s p o n d to the

on t h e vas d e f e r e n s 52'59, the m a j o r target tissure o f the

direct i n t e r a c t i o n of t h e s e h o r m o n e s . A l t e r n a t i v e l y , n e r v e g r o w t h factor ( N G F ) p r o d u c e d in

H G , and the vas d e f e r e n s p r o d u c e s high levels of N G F 23'26 and m R N A c o d i n g for N G F 1"19"26. It is possi-

m a l e sex o r g a n s such as the vas d e f e r e n s , which is a ma-

ble that N G F levels in the vas d e f e r e n s are r e s p o n s i v e

j o r t a r g e t of the h y p o g a s t r i c g a n g l i o n , m a y r e s p o n d to

to castration and that t e s t o s t e r o n e indirectly influences

c h a n g e s in a n d r o g e n levels and m e d i a t e the r e s p o n s e of

T H biosynthesis in the H G .

tyrosine h y d r o x y l a s e in the h y p o g a s t r i c g a n g l i o n in an indirect m a n n e r . N e r v e g r o w t h f a c t o r influences the biosynthesis of T H ; N G F t r e a t m e n t results in i n c r e a s e d levels of T H activity and T H p r o t e i n in the S C G o f newb o r n rats 7 and in o r g a n cultures of S C G 33. A d d i t i o n a l l y , T H is i n d u c e d in a d r e n a l c h r o m a f f i n cells t r e a t e d with N G F 42. T e s t o s t e r o n e alters the p r o d u c t i o n of N G F in the m a l e m o u s e s u b m a x i l l a r y gland 3"24"27 which is a t a r g e t

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Acknowledgements. The authors wish to thank Rlta Lenertz for administrative and secretarial services and Lynne Cousins for technical assistance. The work was supported m part by postdoctoral fellowships NINDS No NS08465 (M.E.G) and NINDS No NS22103-04 (R.W.H) and Monroe Community Hospital/University of Rochester research support to the Neurogerontology Unit.

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