Distribution and postnatal changes of aromatase mRNA in the female rat brain

ft. Steroid Biochem. Molec. Biol. Vol. 48, No. 5/6, pp. 529-533, 1994 Copyright © 1994 Elsevier Science Ltd 0960-0760(93)E0060-K Printed in Great Britain. All rights reserved 0960-0760/94 $7.00 + 0.00

Pergamon

Distribution and Postnatal Changes of A r o m a t a s e m R N A in the F e m a l e R a t B r a i n Naoko Mouri Yamada, Shuji Hirata and Junzo Kato* Department of Obstetrics and Gynecology, Yamanashi Medical University, Shimokato 1110, Tamaho, Nakakoma, Yamanashi, 409-38, Japan D i s t r i b u t i o n a n d p o s t n a t a l c h a n g e s o f a r o m a t a s e m R N A in t h e W i s t a r s t r a i n f e m a l e r a t s b r a i n w e r e i n v e s t i g a t e d to e l u c i d a t e t h e m e c h a n i s m o f t h e r e g i o n - s p e c i f i c a n d s t a g e - r e l a t e d r e g u l a t i o n s o f b r a i n aromatase activity. Total RNAs extracted from the hypothalamus-preoptic area (HPOA), amygdala, c e r e b r a l c o r t e x , c e r e b e l l u m a n d a n t e r i o r h y p o p h y s i s w e r e s u b j e c t e d to a q u a n t i t a t i v e r e v e r s e transcription-polymerase chain reaction-Southern blotting (RT-PCR-SB) assay. The levels of a r o m a t a s e m R N A w e r e as f o l l o w s : a m y g d a l a > c e r e b r a l c o r t e x - H P O A >> c e r e b e l l u m - a n t e r i o r hypophysis. These results roughly paralleled the distribution of the aromatase activity and the aromatase protein reported previously, with the exception of that of the cerebral cortex. The p o s t n a t a l d e v e l o p m e n t a l p a t t e r n s o f a r o m a t a s e m R N A in t h e H P O A a n d c e r e b r a l c o r t e x w e r e f u r t h e r s t u d i e d . T h e l e v e l s o f a r o m a t a s e m R N A in t h e H P O A t e n d e d to be h i g h a r o u n d b i r t h ( d a y 0) a n d to d e c r e a s e at d a y s 4-8 f o l l o w e d b y n o r e m a r k a b l e c h a n g e to a n a d u l t . T h e d e v e l o p m e n t a l p a t t e r n in t h e H P O A w a s e s s e n t i a l l y s i m i l a r to t h a t o f a r o m a t a s e a c t i v i t y . O n t h e o t h e r h a n d , in t h e cerebral cortex where very low or no aromatase activity was reported throughout developmental s t a g e s , a r o m a t a s e m R N A w a s l o w b u t d e t e c t a b l e l e v e l a r o u n d d a y 0, t h e r e a f t e r g r a d u a l l y i n c r e a s e d to a n a d u l t l e v e l . F r o m t h e s e r e s u l t s , it is i n f e r r e d t h a t s y n t h e s i s o f t h e a r o m a t a s e p r o t e i n by t h e l e v e l o f t h e m R N A s e e m s to m a i n l y r e g u l a t e t h e a r o m a t a s e a c t i v i t y in a r e g i o n - s p e c i f i c a n d s t a g e - r e l a t e d f a s h i o n in m o s t p a r t s o f t h e r a t b r a i n , e x c e p t f o r t h e c e r e b r a l c o r t e x w h e r e t h e posttranscriptional regulation may play an important role.

J. Steroid Biochem. Molec. Biol., Vol. 48, No. 5/6, pp. 529-533, 1994

INTRODUCTION

immun'ohistochemical studies in a majority of the brain areas where aromatase activity was present (with a T h e formation of estrogens from androgens, which is few exceptions)[11-14], suggesting that the level of catalyzed by aromatase P - 4 5 0 enzyme, is believed to aromatase protein is strongly associated with the mediate the sexual differentiation of neural structures aromatase activity in the rat brain. Therefore, investiand reproductive behavior[I--4]. Aromatase activity gation of the distribution of the aromatase m R N A in has been detected in several regions of the rat brain. the brain was necessary to elucidate the region-specific T h e level of aromatase activity is highest in the h y p o - and stage-related regulations of aromatase activity. thalamic and limbic areas but there is little or no However, little information on the m R N A was availactivity in the cerebral cortex, cerebellum and anterior able so far. In this context, we attempted to conduct pituitary [5-8]. F u r t h e r m o r e , previous reports on onto- biochemical detection and comparison of aromatase geny of brain aromatase activity [9, 10] revealed that m R N A in the rat brain. Since intensity of the signal the activity in the hypothalamus was the highest at late of aromatase m R N A detected by N o r t h e r n blotting in embryonic stage and high at early postnatal stage our preliminary study was very weak in the amygdala followed by decreasing to an adult level with very (data not shown), which contained the greatest amount low or no activity in the cerebral cortex throughout of aromatase activity in the rat brain [6-10], it indicated all developmental stages. T h e mechanism of these the inadequate sensitivity of N o r t h e r n blotting for region-specific and stage-related regulations of the analyzing the level of the message in the various brain brain aromatase activity has not been clarified yet. regions. In the present study, we employed a quantitatRecently, the aromatase protein was demonstrated by ive reverse transcription-polymerase chain reactionSouthern blotting ( R T - P C R - S B ) assay to investigate distribution and postnatal changes of aromatase m R N A *Correspondence to J. Kato. Received 4 June 1993; accepted 14 Dec. 1993. in the rat brain. 529

Naoko Mouri Yamada et al.

530 MATERIALS AND METHODS

Animals and tissues T o analyze the intracerebral distribution of aromatase m R N A , three groups of 8-week-old diestrous Wistar strain female rats, consisting of 3 rats per group, were killed by decapitation and then the hypothalamus and preoptic area (HPOA), amygdala (AMY), cerebral cortex (CC), cerebellum (Ce), anterior hypophysis (AP) were dissected and removed, as described previously [15]. T o analyze the postnatal changes of aromatase m R N A , female Wistar strain rats at - 2 , 0, 2, 4, 8, 12 and 18 days of age, and at 8-week-old (diestrous) were used. T h e rats were divided by ages, and then subdivided into 3 subgroups per age (n = 5 - 6 / s u b g r o u p for ages: - 2 , 0 and 2 days, n = 3/subgroup for ages: 4, 8, 12, 18 days and 8 weeks). T h e CC and the H P O A were dissected from the rats. In addition, the ovary from 8-week-old diestrous rats was removed for use as a positive control because it has the greatest activity of aromatase.

Chemicals and laboratory equipment All reagents used in these studies were the highest grade available. Water was double distilled and autoclaved, or treated with an additional 0.1% diethylpyrocarbonate and reautoclaved. All equipment was autoclaved and/or sterilized at 200°C to eliminate RNase activity. Microtubes, pipette tips and centrifugation tubes were used only once to avoid contamination with amplified or cloned genes.

R N A extraction T h e tissues were frozen in liquid nitrogen and kept at - 8 0 ° C until R N A extraction. Total R N A was extracted from each tissue by the g u a n i d i u m cesium chloride ultracentrifugation method [16] and the R N A concentration was determined by U V absorption.

Reverse transcription (R T) Total R N A extracted from each tissue was reverse transcribed to synthesize single stranded c D N A . Briefly, various amounts from five brain tissues (to analyze the intracerebral distribution, see below), 20 ng from the H P O A and the CC at each developmental stage (to analyze the ontogeny), and graded diluted ovarian total R N A were incubated at 42°C for 60 min with 5 U of RAV-2 reverse transcriptase (Takara, Kyoto, Japan) in a 25 #1 reaction volume containing 5 0 m M T r i s - H C 1 (pH8.3), 1 0 0 m M KC1, 1 0 m M MgC12, 10 m M dithiothreitol, 1 m M for each d N T P and 1 0 # M random hexadeoxynucleotide primer (Takara, Kyoto, Japan).

Oligonucleotide primers T h e sequences of the oligonucleotide primers for aromatase m R N A are as follows; rAMs: 5 ' - C T G A A CATCGGAAGAATGCACAGGC-3', and rAMas: 5'-ATTTCCACAATGGGGCTGTCCTCAT-3'.

T h e primer set flanked the rat aromatase c D N A sequence from base 1261 to base 1544, as n u m b e r e d by Hickey et al. [17], which was considered to contain a splicing site judging from the h u m a n aromatase gene structure [18]. T h e sequences of the primers for fl actin m R N A are as follows; f l A s : 5 ' - A T C G T G G G C C G C C C T A G G C A - 3 ' , and flAas : 5 ' - T G G C C T T A G G G T T C A G A G G G G - 3 ' . T h e primer set flanked the rat fl actin c D N A sequence from base 100 to base 343, as n u m b e r e d by Nudel et al. [19], which consisted of the first and second exons.

Polymerase chain reaction (PCR) T h e single stranded c D N A was subjected to PCR. Briefly, 1 #1 of c D N A (1/25 of obtained c D N A ) was amplified in a 25/~1 reaction volume containing 50 m M KC1, 1 . 5 m M MgC12, 0.01% (w/v) gelatin, 1 0 m M T r i s - H C 1 (pH 8.3), 200/~M of each d N T P , 10 # M of each primer and 1.25 unit of Taq D N A polymerase (Perkin Elmer Cetus, Norwalk, C T , U.S.A.). T h e reaction was performed for 26 cycles (to analyze aromatase m R N A ) or 18 cycles (to analyze fl actin m R N A ) . Each cycle consisted of an incubation period of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C with the last phase of 72°C of the last cycle extended to 11 min.

Direct nucleotide sequencing T h e R T - P C R product generated from either ovary or H P O A , purified by electrophoresis, was subjected to direct nucleotide sequencing using Taq D N A polymerase according to the dideoxy method of Sanger et al. [20] with modifications.

Southern blotting ( S B ) One microliter of the R T - P C R product (1/25 of obtained product) from each tissue was electrophoresed in a 2.0% agarose gel. After the electrophoresis, the product was transferred onto a nylon m e m b r a n e ( H y b o n d N + , Amersham) using 400 m M N a O H as a transfer solution for 3 h. T h e m e m b r a n e was prehybridized in the prehybridization buffer [6 × SCC (1 × SCC:0.15 M sodium chloride-0.015 M sodium citrate), 1 5 0 # g / m l yeast R N A , 1% sodium dodecil sulfate (SDS)] at 42°C for 3 h. T h e n the m e m b r a n e was hybridized with a 32p labeled rat aromatase or fl actin c D N A probes in the same buffer at 65°C for 12 h. T h e probes were generated from the respective R T P C R products by the random priming method (random priming D N A labeling kit, Takara). After the hybridization, the m e m b r a n e was rinsed twice in 2 × SSC, 1% SDS at room temperature for 10 min and then washed twice in 0.1 × SSC, 1% S D S at 65°C for 20min. T h e hybridization signal was analyzed by a bio-image analyzer, BAS2000 (Fuji Film, Tokyo, Japan).

Comparison of the levels of aromatase m R N A and fl actin mRNA In order to compare the levels of aromatase m R N A and fl actin m R N A , total R N A of 1280 ng from AP and

Aromatase mRNA in Rat Brain Ce, 160 ng from A M Y and CC, 40 ng from H P O A (to analyze the intracerebral distribution of aromatase m R N A ) , 20 ng of the total R N A from five brain regions (to analyze the level of 13 actin m R N A ) , 20 ng from the H P O A and the CC at each developmental stage (to analyze the ontogeny of both m R N A s ) , and graded diluted ovarian total RNAs; 20, 10, 5, 2.5, 1.3, 0.63, 0.32, 0.16, 0.08ng; were simultaneously subjected to R T - P C R - S B . Using the standard curve which was generated from the amount of ovarian R N A s , the level aromatase m R N A in each tissue was calibrated. RT-PCR

blank

W h e n distilled water, used as a R T - P C R blank, was simultaneously subjected to the R T - P C R - S B with the same reagents, no specific signal could be obtained, indicating that no contamination of any reagents occurred in these experiments. Statistics Data were expressed as the m e a n + SD of the 3 groups (n = 3/group). A paired Student's t-test was used to test for statistical significance of difference between tissues.

R E S U L T S AND D I S C U S S I O N The R T - P C R product from the rat brain T h e R T - P C R product of 2 8 4 b p , which corresponded in length to the distance between the 5/ends of the two primers on the rat aromatase c D N A , was generated from the five regions of the brain and ovary. Since the amplified region contained a splicing site (which was predicted by comparison with the aromatase gene structure of the h u m a n [18]), the product of 2 8 4 b p could originate from aromatase m R N A in these tissues and not from genomic D N A . T w o slightly different sequences of the rat aromatase c D N A s were reported; the c D N A sequence derived from the ovarian granulosa cells was reported by Hickey et al. [17], and the sequence from the Leydig cell t u m o r line was reported by L e p h a r t et al. [18]. T h e nucleotide sequence of the R T - P C R product in this study was identical to the former one, implying that mutations of the aromatase gene might occur in the t u m o r cells. F u r t h e r m o r e , it was reported that three different aromatase m R N A s were expressed in the rat ovary; only the largest m R N A species (at 2.7 kb) appeared to be functional and the two smaller species could not encode a functional aromatase as they lacked the sequence corresponding to the heine-binding domain [21]. However, the R T P C R product in this study could only originate from the "functional" m R N A and not from the " n o n functional" m R N A species because the primers for aromatase m R N A flanked the region including the h e m e - b i n d i n g domain corresponding sequence of the cDNA.

531

A standard curve for comparison of the levels of aromatase m R N A A standard curve was prepared from the radioactivity of the signals of the R T - P C R products generated from the graded diluted control R N A s (data not shown). Duplicate assays, which resulted in a highly reproducible response pattern, showed a linear correlation between the logarithmic value of the radioactivities of the product signals and that of the weight of the template R N A within the range between 10 and 0.32 ng of its starting weight. Using the standard curve, the level of rat aromatase m R N A could be estimated in different tissues and developmental stages. The intracerebral distribution of rat aromatase m R N A T h e levels of aromatase m R N A were as follows: A M Y > C C - H P O A >> C e - A P (Fig. 1), which roughly paralleled the level of aromatase activity and aromatase protein with the exception of C C [5-8, 11-14]. T h e s e findings suggest that the level of the aromatase m R N A mainly regulated aromatase activity in a region-specific fashion through the regulation of synthesis of aromatase protein, in the most of the rat brain regions. It should be noted that a relatively high level of aromatase m R N A was detected in CC where the level of aromatase activity and aromatase protein were reported to be little or absent [5-8, 11-14]. The postnatal changes of the aromatase m R N A in the rat brain T h e postnatal developmental patterns of aromatase m R N A in the H P O A and C C were further studied to elucidate the mechanism of the stage-related

q)

(%) 150

E o>. --¢100

o<

~z

~no E

E

50

11.

AP

HPOA

AMY

CC

Ce

Fig. 1. T h e i n t r a c e r e b r a l d i s t r i b u t i o n o f r a t a r o m a t a s e m R N A . T o t a l R N A s o f 1280 n g f r o m A P a n d Ce, 160 n g f r o m A M Y a n d CC, 40 n g f r o m H P O A w e r e s u b j e c t e d to a q u a n t i tative RT-PCR assay. The level of aromatase mRNA was calculated using the standard curve and the mean level of aromatase mRNA obtained in the AMY was assigned a value o f 100%. E a c h v a l u e r e p r e s e n t s t h e m e a n a n d S D ( v e r t i c a l bar) of 3 groups. HPOA: hypothalamus and preoptic area A M Y : a m y g d a l a , CC: c e r e b r a l c o r t e x , Ce: c e r e b e l l u m , A P : anterior hypophysis, RT-PCR: reverse transcriptionpolymerase chain reaction.

532

Naoko Mouri Yamada et al. 0 0.-~

(%)

!~ -

i

150

_c~

'0

c

d

100

50

0~.., ft.

I -2

I 0

I 2

I 4

I 8

at birth

I 12

I 18 days of age

'/"

I 8

weeks of age

Fig. 2. T h e o n t o g e n y o f a r o m a t a s e m R N A in t h e H P O A ( d a s h e d line) a n d t h e C C (solid line). 20 ng f r o m t h e H P O A a n d t h e C C at e a c h d e v e l o p m e n t a l s t a g e w e r e s u b j e c t e d to a q u a n t i t a t i v e R T - P C R a s s a y . T h e m e a n level of a r o m a t a s e m R N A o b t a i n e d in t h e H P O A o f t h e 2 - d a y - o l d r a t ( d a s h e d line) a n d C C o f t h e 8 - w e e k - o l d r a t (solid line) w a s a s s i g n e d a v a l u e o f 100%, r e s p e c t i v e l y . E a c h v a l u e r e p r e s e n t s t h e m e a n a n d S D ( v e r t i c a l b a r ) o f 3 g r o u p s . H P O A : h y p o t h a l a m u s a n d p r e o p t i c a r e a , CC: c e r e b r a l c o r t e x , a: s i g n i f i c a n t l y l o w e r ( P < 0.01), b: s i g n i f i c a n t l y l o w e r ( P < 0.05), a n d c: s i g n i f i c a n t l y h i g h e r ( P < 0.01) t h a n t h e l e v e l o f t h e m R N A in t h e C C o f t h e 8 - w e e k - o l d rat. d: s i g n i f i c a n t l y h i g h e r ( P < 0.01) t h a n t h e level o f t h e m R N A in t h e H P O A of the 8-week-old-rat.

regulations of brain aromatase activity, especially to analyze the developmental changes in dissociation between the levels of the message and protein in the CC. T h e levels of aromatase m R N A in the H P O A tended to be high around birth (day 0) and to decrease at days 4-8 followed by no remarkable change to an adult (Fig. 2, dashed line). T h e aromatase activity in the hypothalamus was reported to be the highest at late embryonic stage (embryonic days 18-20) and high at early postnatal stage followed by gradually decreasing to an adult level [9, 10]. O u r data on the postnatal changes in the m R N A in the tissue was essentially similar to that of aromatase activity. On the other hand, in the C C where very low or no aromatase activity was reported throughout all developmental stages [9, 10], a very low but detectable level of aromatase m R N A was found at 2 days before birth. T h e n the cortical massage level was gradually increased to an adult level (Fig. 2, solid line). In order to validate the R N A concentrations which were determined by U V absorption, the levels of fl actin m R N A in the same R N A were analyzed. W h e n the levels of aromatase m R N A were calibrated by the respective levels of/3 actin m R N A , the postnatal changes of the calibrated aromatase m R N A in both tissues were essentially similar to those without calibration (data not shown). T h u s the postnatal developing patterns of aromatase m R N A indicated in Fig. 2 were considered to reflect true changes in the level of the message. F r o m these results, it was indicated that the postnatal developmental changes in the level of aromatase

activity in the H P O A might be caused by the changes in its message level. On the other hand, in the CC, mismatch between the level of aromatase activity and aromatase m R N A increased with development, implying that the posttranscriptional regulation might play an important role in the tissue. Aromatase m R N A

in the C C

Dissociation of the levels between aromatase m R N A and aromatase protein may not be only specific to CC, because mismatch of the levels of the m R N A and the protein has been also reported in the rat corpus luteum at mid-gestation [22-24]. Possible explanations for the mismatch in C C may be; (1) " n o n - f u n c t i o n a l " m R N A species containing heme-binding domain corresponding sequence are specifically expressed in the CC, (2) specific inhibitory factors of translation of aromatase m R N A is present in C C [25-32], (3) aromatase proteins are inactivated by other enzymes [33]. In addition, it was reported that a mismatch of the levels between aromatase activity and aromatase protein was found in several brain regions, such as the medial preoptic area and the bed nucleus stria terminals [14]. Although reasons for the divergent relationship a m o n g the level of the m R N A , the protein and the activity remain unclear, these mismatches indicate that the region-specific and stage-related regulation of aromatase activity may also involve multiple factors which are not yet identified especially in the CC. Further study on the mechanism of gene expression of aromatase is essential to elucidate biophysical role of a gradual

Aromatase m R N A in Rat Brain

increase in the level o f aromatase m R N A from a n e o n a t e to an adult.

in the C C

Acknowledgements--The authors express appreciation to Ms. Michiko Yoneyama for skillful technical assistance. This work was supported in part by a Grant from the Ministry of Education, No. 01440069, to JK.

REFERENCES

1. Toran-Allerand C. D.: Sex steroid and the development of the newborn mouse hypothalamus and preoptic area in vivo; implications for sexual differentiation. Brain Res. 106 (1976) 407-412. 2. (3orski R. A.: Hormone-induced sex differences in hypothalamic structure. Bull. Tokyo Metro. Inst. Neurosci. 3 (Suppl.) (1988) 67-90. 3. M c E w e n B. S., Leiberburg I., Chaptal C. and Krey L. C.: Aromatization; important for sexual differentiation of the neonatal rat brain. Horm. Behav. 9 (1977) 249-263. 4. Parsons B., Rainbow T. C. and M c E w e n B. S.: Organizational effects of testosterone via aromatization on feminine reproductive behavior and neural progestin receptors in rat brain. Endocrinology 114 (1984) 192-200. 5. Naftolin F., Ryan K. J., Davies I. J., Reddy V. V., Flores F., Petro Z., K u h n M., White R. J., Takaoka Y. and Wolin L.: T h e formation of estrogens by central neuroendocrine tissues. Recent Prog. Horm. Res. 31 (1975) 295-314. 6. SelmanoffM. K., Brodkin L. D., Weiner R. I. and Siiteri P. K.: Aromatization and 5a-reduction of androgens in discrete hypothalamic and limbic regions of the male and female rat. Endocrinology 101 (1977) 841-848. 7. Roselli C. E., Ellinwood W. N. and Resko J. A.: Regulation of brain aromatase activity in rats. Endocrinology 114 (1984) 192-200. 8. Takahashi Y. and Yamanaka H.: T h e regulation system of brain aromatase activity; Distribution and changes with age of aromatase activity in rat brain. Folia Endocr. 63 (1987) 863-869. 9. George F. W. and Ojeda S. R.: Changes in aromatase activity in the rat brain during embryonic, neonatal, and infantile development. Endocrinology 111 (1982) 522-529. 10. T o b e t S. A., Baum M. J., Shim J. H. and Canic C. A.: Aromatase activity in the perinatal rat forebrain: Effects of age, sex and intrauterine position. Devl Brain Res. 23 (1985) 171-178. 11. Shinoda K., Yagi H., Fujita H., Osawa Y. and Shiotani Y.: Screening of aromatase-containing neurons in rat forehrain; an immunohistochemical study with antibody against human placental antigen X-P2 (hPAX-P2). ft. Comp. Neurol. 290 (1989) 502-515. 12. Shinoda K., Yagi H., Fujita H., Osawa Y. and Shiotani Y.: Involvement of specific placental antigen X-P2 in rat olfaction; an immunohistochemical study in the olfactory bulb..7. Comp. Neurol. 294 (1990) 340-344. 13. Balthazart, J., Foidart A., Surlemont C. and Harada N.: Distribution of aromatase-immunoreactive cells in the mouse forebrain. Cell Tissue Res. 263 (1991) 71-79. 14. Sanghera M. K., Simpson E. R., McPhaul M. J., Kozlowski G., Conley A. J. and Lephart E. D.: Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide generated polyclonal antibodies. Endocrinology 129 (1991) 2834-2844. 15. Kato J. and Villee C. A.: Preferential uptake of estradiol by the anterior hypothalamus of the rat. Endocrinology 80 (1966) 567-575. 16. Aviv H. and Leder P.: Purification of biologically active gloin messenger R N A by chromatography on oligothymidylic acid cellulose. Proc. Nam. Acad. Sci. U.S.A. 69 (1972) 1408-1412.

533

17. Hickey G. J., Krasnow J. S., Beattie W. G. and Richard J. S.: Aromatase cytochrome P450 in rat ovarian granulosa cells before and after luteinization; Adenosine 3', 5'-monophosphatedependent and independent regulation. Cloning and sequencing of rat aromatase c D N A and 5'genomic DNA. Molec. Endocr. 4 (1990) 3-12. 18. Lephart E. D., Peterson K. (3., Noble J. F., George F. W. and McPhaul M. J.: T h e structure of c D N A clones encoding the aromatase P-450 isolated from a rat Leidig cell tumor line demonstrates differential processing of aromatase m R N A in rat ovary and a neoplastic cell line. Molec. Cell. Endocr. 70 (1990) 31-40. 19. Nudel U., Zakut R., Shani M., Neuman S., Levy Z. and Yaffe D.: T h e nucleotide sequence of the rat cytoplasmic fl actin gene. Nucleic Acids Res. 11 (1983) 1579-1771. 20. Sanger F., Nicklin S. and Coulson A. R.: D N A sequencing with chain terminating inhibitors. Proc. Natn. Acad. Sci. U.S.A. 74 (1977) 5463-5467. 21. Lephart E. D., Simpson E. R. and McPhaul M. J.: Ovarian aromatase cytochrome P-450 m R N A levels correlate with enzyme activity and serum estradiol levels in anestrous, pregnant and lactating rats. Molec. Cell. Endocr. 85 (1992) 205-214. 22. Richards J. S., Hickey (3. J., Chen S., Shively J. E., Hall P. F., Gaddykurten D. and Kurten R.: Hormonal regulation of estradiol biosynthesis, aromatase activity and aromatase m R N A in rat ovarian follicles and corpora lutea. Steroids 50 (1987) 393-409. 23. Hickey G. J., Chen S., Besman M. J., Shively J. E., Hall P. F., Gaddykurten D. and Richards J. S.: Hormonal regulation, tissue distribution, and content of aromatase cytochrome P450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea; relationship to estradiol biosynthesis. Endocrinology 122 (1988) 1426-1436. 24. Hickey (3. J., Oonk R. B., Hall P. F. and Richards J. S.: Aromatase cytochrome P450 and cholesterol side-chain cleavage P450 in corpora lutea of pregnant rat; diverse regulation by peptide and steroid hormones. Endocrinology 125 (1989) 1673-1682. 25. Mendelson C. R., Corbin C. J., Smith M. E., Smith J. and Simpson E. R.: Growth factors suppress and phorbol esters potentiate the action of dibutyryl adenosine 3', 5' monophosphate to stimulate aromatase activity of human adipose stromal cells. Endocrinology 118 (1986) 968-973. 26. Adashi E. Y. and Resnick C. E.: Antagonistic interactions of transforming growth factors in the regulation of granulosa cell differentiation. Endocrinology 119 (1986) 1879-1881. 27. Fortune J. E., Wissler R. N. and Vincent S. E.: Prolactin modulates steroidogenesis by rat granulosa cell: Effects of estradiol. Biol. Reprod. 35 (1986) 92-99. 28. Krasnow J. S., Hickey (3. J. and Richards J. S.: Regulation of aromatase m R N A and estradiol biosynthesis in rat ovarian granulosa and luteal cells by prolactin. Molec. Endocr. 4 (1990) 13-21. 29. Hsueh A. J. W. and Erickson.(3. F.: Glucocorticoid inhibition of FSH-induced estrogen production in cultured rat granulosa cell. Steroids 32 (1978) 639--647. 30. Schonmaker J. N. and Erickson G. F.: Glucocorticoid modulation of follicle-stimulating hormone-mediated granulosa cell differentiation. Endocrinology 113 (1983) 1356-1363. 31. Schreibar J. R., Nakamura K. and Erickson G. F.: Progestins inhibit FSH-stimulated granulosa estrogen production at a postcAMP site. Molec. Cell. Endocr. 21 (1981) 161-170. 32. Fortune J. E. and Vincent S. E.: Progesterone inhibits the induction of aromatase activity in rat granulosa cell in vitro. Biol. Reprod. 28 (1983) 1078-1089. 33. Hickey G. J., Chen S., Besman M. J., Shively J. E., Hall P. F., Gaddy-Kurten D. and Richards J. S.: Hormonal regulation, tissue distribution, and content of aromatase cytochrome P-450 messenger ribonucleic acid and enzyme in rat ovarian follicles and corpora lutea: Relationship to estradiol biosynthesis. Endocrinology 122 (1988) 1426-1436.