Molecular Brain Research, 16 (1992) 187-192

187

© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00

BRESM 70533

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat Edwin

D. Lephart

a,b,d

Evan

R. Simpson

Jean

D. Wilson

a,b,d

Michael

J. M c P h a u l

c and Sergio R. Ojeda

c, M i c h a e l

W. Kilgore

a,b,d

e

a The Cecil H. and Ida Green Center for Reproductive Biology Sciences and The Departments of b Biochemistry, c Internal Medicine, and d Obstetrics and Gynecology, The University of Texas, Southwestern Medical Center, Dallas, TX 75235 (USA) and e Dit,ision of Neuroscience, Oregon Regional Primate Research Center, Beacerton, OR 97006 (USA)

(Accepted 11 August 1992)

Key words: Aromatase; Brain; Prenatal; Development; Rat

Aromatase cytochrome P-450 (P-450AROM)enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 ( < 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 ( > 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (= 1 pmol/h/mg protein). The profile of P-450AROMmRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROMmRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROMmRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROMmRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels. These findings provide evidence for the presence of P-450AROMmRNA in neural tissue and suggest that increases in P-450AROMmRNA levels determine, at least in part, enzymatic activity during the period of development where brain aromatase is increasing. In contrast, the perinatal decline in enzyme activity appears to be regulated by factors that operate via mechanisms other than inhibition of P-450AROMgene expression.

INTRODUCTION It is well e s t a b l i s h e d t h a t t h e c e n t r a l n e r v o u s system is able to a r o m a t i z e a n d r o g e n s to e s t r o g e n s 8'9'35. A l t h o u g h the r a t e s o f a r o m a t a s e activity in t h e m a m m a l i a n b r a i n a r e low c o m p a r e d to t h o s e in t h e ovary 45 a n d p l a c e n t a 1, t h e activity is d e t e c t a b l e in h y p o t h a l a mic a n d limbic a r e a s t h r o u g h o u t d e v e l o p m e n t , e s p e cially d u r i n g t h e p r e n a t a l p e r i o d 15,26,29'49. E s t r o g e n s f o r m e d locally in t h e b r a i n a r e t h o u g h t to b e involved in the sexual d i f f e r e n t i a t i o n o f n e u r a l s t r u c t u r e s 17'47'48, the r e g u l a t i o n o f r e p r o d u c t i v e f u n c t i o n 31'3s a n d sexual

behavior 14,38 D e t e c t i o n o f n e u r a l a r o m a t a s e involves m e a s u r i n g the r a t e o f e n z y m e activity by e i t h e r e s t r o g e n p r o d u c t

isolation using thin layer c h r o m a t o g r a p h y 24 or by t h e r e l e a s e of ' t r i t i a t e d w a t e r ' f r o m r a d i o l a b e l e d p r e cursors 46. Since t h e p r e s e n c e o f t h e e n z y m e in b r a i n was first d e s c r i b e d 36'37, c o n s i d e r a b l e a t t e n t i o n has b e e n given to its c h a r a c t e r i z a t i o n 8'9't5'26'29'35'4°'49, b u t the factors involved in r e g u l a t i n g n e u r a l a r o m a t a s e activity r e m a i n p o o r l y u n d e r s t o o d 7'1°'26'41'42. A c D N A e n c o d i n g t h e rat a r o m a t a s e c y t o c h r o m e P-450 (P-450AROM) has b e e n i s o l a t e d in o n e o f o u r l a b o r a t o r i e s from a L e y d i g cell t u m o r line 23. U s i n g this c D N A , t h r e e species o f P-450AROM m R N A w e r e det e c t e d in a d u l t r a t o v a r i a n tissue (i.e. 1.7, 2.2 a n d 2.7 kb), c o n f i r m i n g a p r e v i o u s r e p o r t 18. F u r t h e r analysis r e v e a l e d t h a t the two s m a l l e r m R N A species lack t h e h e m e - b i n d i n g d o m a i n a n d thus p r e s u m a b l y e n c o d e

Correspondence: E.D. Lephart, Green Center, Y6.316, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard,

Dallas, TX 75235-9051, USA. Fax: (1) (214) 688-8683.

188 nonfunctional aromatase proteins. Hence, the functional rat P-450A~tO M in ovarian tissue appears to be encoded by the 2.7 kb aromatase m R N A spectes ~ . In the present study, we compared the ontogeny of aromatase activity in the rat brain (using the tritiated water assay) with that of rat aromatase P-450AROM m R N A levels (using R N A blot hybridization) to determine whether the marked changes in aromatase activity observed in vivo during late fetal and perinatal development are accompanied by corresponding changes in P-450AROM m R N A levels. •

MATERIALS

")~,

AND METHODS

Materials [l/3,2/3-3H]Testosterone (T) (42 C i / m m o l ) was purchased from New England Nuclear Corp. (Boston, MA). [1/3-3H]T was prepared as previously described27; the final specific activity was 20.1 C i / m m o l . The sources of all other supplies and reagents are detailed elsewhere ~5.21.22,23.26.27. Animals Time-mated pregnant rats (sperm positive date = day 0 of gestation) were obtained from Holtzman Co. (Madison, WI) and were housed in a controlled environment (lights on 07.00-19.00 h, temperature 23-25°C) with free access to tap water and rat laboratory chow (Teklad Rat Diet; Madison, WI). At each indicated gestational day (GD), the pregnant rats were killed by decapitation (between 10.00 and 14.00 h) and tissue fragments containing the medial basal hypothalamus-preoptic area ( M B H - P O A ) were collected from male and female fetuses, as previously described ~s'26. Aromatase actiuity To assess aromatase activity in hypothalamic tissue fragments the "tritiated water-release assay' was used. This method has been described in detail previously 22'24'26'27, therefore, only a brief description is provided below. Tritiated water-release assay In preliminary experiments, M B H - P O A tissue fragments were incubated for 1 h at 37°C in 0.2 ml Dulbecco's modified Eagle medium (DMEM), pH 7.4, containing [1/3-3H]T in various concentrations under an atmosphere of 95% 0 2 / 5 % C02. The incubations were terminated by the addition of 1 ml chloroform to each tube, and the 3 H 2 0 released was quantified by counting an aliquot of the aqueous phase (1 ml) in 10 ml of Picofluor scintillation cocktail. Maximal rates of activity were obtained at a substrate concentration of 0.3 /zM [I/3-3H]T using day 19 fetal neural tissue (Fig. 1). As previously demonstrated, this activity was inhibited ( > 80%) by 0.25 /xM 4-hydroxyandrostenedione, a known aromatase inhibitor (data not shown) ~s26. We also determined that aromatase activity in MBHPOA is linear with time (up to 90 rain) and increasing amounts of tissue (up to 1 mg; data not shown). The protein content of each tissue fragment was determined by the method of Lowry et al. 28. The results are expressed as aromatase activity in p m o l / h / m g protein. Standard assay conditions were adopted in which tissue fragments were incubated for 1 h at 37°C in D M E M containing a saturating concentration (0.3 ~ M ) of [1/3-3H]T. RNA isolation and preparation of poly(A)-enriched RNA Fetal MBH-POA, adult ovarian tissue from pregnant animals at gestational day (GD 18) and adult female liver were dissected, frozen immediately on dry ice and stored at - 7 0 ° C until R N A isolation. For M B H - P O A samples, the tissue was collected from fetuses of three pregnant rats for each gestational age. Total R N A was pre-

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