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Preferential uptake of 6, 7-H3-estradiol by the endometrium of the rat uterus
737
PREFERENTIAL
UPTAKE
O F 6, 7 - H 3 - E S T R A D I O L B Y T H E
ENDOMETRIUM
OF THE RAT UTERUS~
/ames
W. F l e s h e r
D e p a r t m e n t of P h a r m a c o l o g y U n i v e r s i t y of K e n t u c k y Lexington, Kentucky
Received February
i0, 1965
ABSTRACT
T h e u p t a k e of t r i t i a t e d e s t r a d i o l b y the e n d o m e t r i u m a n d m y o m e t r i u m of the r a t u t e r u s h a s b e e n s t u d i e d f o l l o w i n g i n t r a venous administration. These data demonstrate the p r e f e r e n t i a l u p t a k e of r a d i o a c t i v i t y b y t h e e n d o m e t r i u m . Preferential u p t a k e b y the e n d o m e t r i u m m a y e x p l a i n the g r e a t e r s e n s i t i v i t y of t h e e n d o r n e t r i u r n to c h a n g e s in the l e v e l s of c i r c u l a t i n g e s t r o gens.
Previous
studies have shown that uterus and vagina are able
to t a k e up a n d r e t a i n m o r e of t r i t i a t e d e s t r a d i o l t h a n o t h e r t i s s u e s of t h e i m m a t u r e
r a t f o l l o w i n g s y s t e m i c a d m i n i s t r a t i o n ( i p Z).
Ear-
l l e r , i t w a s s h o w n t h a t h e x e s t r o l of h i g h s p e c i f i c a c t i v i t y r e a c h e s a maximum
l e v e l in the u t e r u s of i m m a t u r e
hours after injection(3).
More recently,
goats and sheep 2-6
a similar incorporation
p a t t e r n h a s b e e n f o u n d in the o v a r i e c t o m i z e d
mouse following
intravenous and subcutaneous administration
of t r i t l a t e d e s t r a d i o l
a n d e s t r o n e ( 4, 5).
Using autoradiography,
p h y s i o l o g i c a l a m o u n t s of t r i t i a t e d e s t r a d i o l
mice given hyperw e r e f o u n d to
738
S T E R O I D S
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a c c u m u l a t e r a d i o a c t i v i t y in the u t e r i n e e n d o m e t r i u m ( 6 ) . T h e p r e s e n t p a p e r r e p o r t s on the u p t a k e of t r i t l a t e d e s t r a d i o l by t h e u t e r i n e e n d o m e t r l u r n a n d m y o m e t r i u r n of the y o u n g a d u l t rat~ w h e n a d m i n i s t e r e d in a p h y s i o l o g i c a l d o s e .
M ~ T E R . I A L S AND M E T H O D S
S o l u t i o n s : 6, 7 - H 3 - e s t r a d i o l - 1 7 B ( s p e c i f i c a c t i v i t y 3 6 . 8 c u r i e s / r n l i L i m o l e ) was p u r c h a s e d f r o m New E n g l a n d N u c l e a r C o r p , It w a s p u r i f i e d by d e s c e n d i n g p a p e r c h r o m a t o g r a p h y in a m o d i f i e d B u s h B - 3 s y s t e m : H e p t a n e 333 rnl, b e n z e n e I()7 m l , m e t h a n o l 400 rnl, w a t e r I00 m l . C o n t r o l s p o t s of n o n - r a d i o a c t i v e s t e r o i d w e r e d e t e c t e d on p a p e r c h r o m a t o g r a m s a f t e r d i p p i n g the c h r o m a t o g r a m s in a m i x t u r e of I% F e C I 3 a n d I% K F e (CN)6 a n d t h e w a s h i n g w i t h 6NHCI a n d f i n a l l y w i t h w a t e r . T h e z o n e c o r r e s p o n d i n g to c o n t r o l e s t r a d i o l w a s e l u t e d w i t h m e t h a n o l . T h e p u r i f i e d m a t e r i a l w a s e v a p o r a t e d w i t h N2, w i t h g e n t l e w a r m i n g , to a s m a l l v o l u m e a n d d i l u t e d w i t h b e n z e n e . R ~ d i o c h e r n i c a l p u r i t y was checked by a n a l y t l c a l p a p e r c h r o m a t o g r a p h y as follows: A f t e r d e v e l o p m e n t w i t h t h e s o l v e n t , ( B u s h B - 3 s y s t e m ) the c h r o r n a t o g r a m w a s d r i e d in a i r , c u t i n t o 1 c m s t r i p s a n d e l u t e d i n d i v l d u a l l y in c o u n t i n g b o t t l e s w i t h 5 m l of s p e c t r o g r a d e d i o x a n e . A f t e r s t a n d i n g o v e r n i g h t , I0 rnl of P p O ( 1 3 ) - t o l u e n e (4 g / l i t e r ) w a s a d d e d a n d t h e s a m p l e s w i t h the s t r i p s s t i l l in p l a c e w e r e c o u n t e d in t h e T r i - C a r b Liquid S c i n t i l l a t i o n S p e c t r o m e t e r . M o r e t h a n 96 p e r c e n t of t h e r a d i o a c t i v i t y w h i c h o c c u r r e d in a s i n g l e p e a k w a s f o u n d to be 6, 7 - H 3 - e s t r a d i o l - 1 7 B . P o r t i o n s of the m e t h a n o l - b e n z e n e s o l u t i o n w e r e e v a p o r a t e d w i t h N 2 to n e a r d r y n e s s . A b s o l u t e e t h a n o l w a s t h e n a d d e d a n d e v a p o r a t e d to n e a r d r y n e s s . T h e p r o c e d u r e w a s r e p e a t e d a n d the f i n a l s a l i n e s o l u t i o n ( 0 . 9 % N a C I ; I% e t h a n o l ) w a s m a d e up to c o n t a i n t h e s t e r o i d in a c o n c e n t r a t i o n of 0, 4 ~ g / m l , s p e c i f i c a c t i v i t y 100~c/ug" A n i m a l s : F e m a l e S p r a g u e - D a w l e y r a t s , 2 0 5 - 2 2 4 g, w e r e Lightly a n e s t h e t i z e d w i t h e t h e r a n d t h e s a p h e n o u s v e i n e x p o s e d f o r i n t r a v e n o u s i n j e c t i o n . T h e a n i m a l s w e r e s a c r i f i c e d by d e c a p i t a t i o n two h o u r s a f t e r t h e i n t r a v e n o u s a d m i n i s t r a t i o n of 0. l~g of b, 7 - H 3 - e s t r a d i o l p e r I00 g b o d y w e i g h t , u t e r i q u i c k l y removed and dissected from surrounding tissue. Each uterine horn was opened with a longitudinal cut and
May 1965
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739
l a i d on a g l a s s s l i d e . T h e e n d o m e t r z u m t h u s e x p o s e d w a s s c r a p e d off w i t h t h e e d g e of a s e c o n d g l a s s s l i d e . H i s t o l o g i c a l e x a m i n a t i o n ( t i s s u e e m b e d d e d in p a r a f f i n , s e c t i o n s s t a i n e d with picric acid, acid fuchsin and hematoxylin) indicated that the p r o c e d u r e r e m o v e d a b o u t 75% of the e n d o m e t r i u r n . T h e e n d o m e t r i a l s a m p l e c o n t a i n e d no m y o m e t r i a l e l e m e n t s . I m m e d i a t e l y f o l l o w i n g s e p a r a t i o n of the t i s s u e s , t h e y w e r e t r a n s f e r r e d to t a r e d w e i g h i n g b o t t l e s . A f t e r w e i g h i n g , the s a m p l e w a s d r i e d a t h i g h v a c u u m a n d r e w e i g h e d . R a d i o A s s a y : C o m b u s t i o n s of d r i e d e n d o m e t r i u m , m y o m e t r i u r n a n d b l o o d w e r e c a r r i e d o u t by the o x y g e n - f l a s k t e c h n i q u e u s i n g a m o d i f i c a t i o n - of the m e t h o d of K a l b e r e r a n d R u t s c h m a n (7). S p e c t r o g r a d e d i o x a n e (11. O ml) w a s t r a n s f e r r e d i n t o the w e U of 1 l i t e r c o m b u s t i o n f l a s k . T h e d i o x a n e w a s f r o z e n by a c e t o n e - d r y - i c e m i x t u r e a n d the f l a s k c h a r g e d w i t h pure oxygen. Combustion was started with a projection lamp ( S y l v a n i a , T r u - F l e c t o r , 150 w a t t s ) . T h e d i o x a n e w a s m e l t e d by i m m e r s i n g the w e l l in w a r m w a t e r a n d the f l a s k r o t a t e d e n d o v e r e n d to i n s u r e c o m p l e t e m i x i n g a s p r e v i o u s l y d e s c r i b e d (7). A 10 m l p o r t i o n w a s w i t h d r a w n a n d a d d e d to 5 m l of phosphor consisting of 7.5 g P P O , 75 m g P O P O P , iZ0 g napthalene in 500 m l of xylene. Radioactivity was m e a s u r e d in a T r i - C a r b l i q u i d S c i n t i l l a t i o n S p e c t r o m e t e r w i t h a c o u n t i n g e f f i c i e n c y of Z5%.
RESULTS
AND
DISCUSSION
A s s h o w n in T a b l e I, two h o u r s a f t e r the i n t r a v e n o u s a d m i n i s t r a t i o n of 6, 7 - H 3 - e s t r a d l o l ,
t h e c o n c e n t r a t i o n of
r a d i o a c t i v i t y in e n d o m e t r i u r n is a b o u t two a n d o n e - h a l f t i m e s the c o n c e n t r a t i o n in m y o m e t r i u m .
A t the s a m e t i m e , the
l e v e l of r a d i o a c t i v i t y in b l o o d i s o n l y a p p r o x i m a t e l y 5-10% of the l e v e l in u t e r u s . T h e s e d a t a d e m o n s t r a t e the s e l e c t i v i t y of t h e e n d o m e t r i u m in i t s a b i l i t y to a c c u m t t l a t e r a d i o a c t i v i t y .
A l t h o u g h no a t t e m p t
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5:5
Table I
C o m p a r i s o n of l i q u i d - s c i n t i l l a t i o n c o u n t i n g of e n d o m e t r i u m a n d m y o m e t r i u r n o x i d i z e d by the o x y g e n - f l a s k t e c h n i q u e . Tissue
Relative Specific Activity
Endometriurn Myometrium RSA
=
% Wate r
228 + 16 92 + 12 counts/min/mg counts/min/mg
78-80 77-82
tissue whole uterus
x
100
V a l u e s e x p r e s s e d a s r e l a t i v e s p e c i f i c a c t i v i t i e s (RSA) a r e the m e a n + s t a n d a r d e r r o r f o r 6 d e t e r m i n a t i o n s .
w a s m a d e in t h e s e e x p e r i m e n t s to i d e n t i f y the r a d i o a c t i v i t y , i t h a s b e e n p r e v i o u s l y s h o w n t h a t o n l y e s t r a d i o l c o u l d be i s o l a t e d f r o m the i m m a t u r e
r a t u t e r u s ( 1, Z).
It i s t h e r e f o r e r e a s o n a b l e to a s s u m e the r a d i o a c t i v i t y o b s e r v e d is due to 6, 7 - H S - e s t r a d i o l - 1 7 B .
T h e f i n d i n g of
d i f f e r e n t c o n c e n t r a t i o n s of r a d i o a c t i v i t y in e n d o m e t r i u m s u g g e s t several possibilities which could account for these results; (I) a g r e a t e r n u m b e r of r e c e p t o r b i n d i n g s i t e s in e n d o m e t r i u r n than myometrium;
(Z) a m e c h a n i s m f o r the a c c u m u l a t i o n of
h o r m o n e in t a r g e t t i s s u e s ; (3) a h i g h w a t e r c o n t e n t in e n d o metrium,
w h i c h on d r y i n g the t i s s u e r e s u l t s in a h i g h s p e c i f i c
activity.
T h e t h i r d p o s s i b i l i t y w a s e x c l u d e d by t h e f i n d i n g t h a t the
w a t e r c o n t e n t i s the s a m e in e n d o m e t r i u m a n d m y o m e t r i u m ( T a b l e I).
A c c o r d i n g to A s t w o o d (8) t h e p e r c e n t a g e of w a t e r in
M a y 1965
S T E R O ID S
741
w h o l e u t e r u s d u r i n g t h e e s t r o u s c y c l e r a n g e s f r o m 7 9 . 8 to 84.8.
E v i d e n c e f o r t h e p r e s e n c e of r e c e p t o r s h a s b e e n p r e -
s e n t e d in s t u d i e s on t h e s u b c e U u l a r l o c a l i z a t i o n of 6, 7 - H 3e s t r a d i o 1 - 1 7 B in t h e u t e r u s of the o v a r i e c t o m / z e d r a t (9). hal/the
About
r a d i o a c t i v i t y w a s f o u n d a s s o c i a t e d w i t h t h e 105, 000 g
supernatant fraction, which apparently contains a specific "receptor"
substance(s) for estradlol.
s u c h a s l u n g do n o t b i n d e s t r a d i o l ,
Non-target tissues
evidently b e c a u s e the
a p p r o p r i a t e r n a c r o m o l e c u l e s a r e n o t p r e s e n t in l u n g t i s s u e . I t h a s b e e n p o s t u l a t e d by F i s h m a n t h a t t h e g l u c o s i d u r o n i c a c i d of e s t r a d i o l i s f o r m e d in t h e l i v e r a n d i n t e s t i n e a n d i s c a r r i e d to t h e " t a r g e t o r g a n " w h e r e B - g l u c u r o n i d a s e t r a n s f e r s t h e g l u c u r o n y 1 g r o u p to a s u i t a b l e a c c e p t o r a n d r e l e a s e s estradiol(10).
Evidence has also been presented that B-glucur-
o n i d a s e is p r e s e n t i n - h i g h e r c o n c e n t r a t i o n in e n d o m e t r i u m t h a n in m y o m e t r i u m ( 1 1 ) .
A c c o r d i n g to t h i s m o d e l , one w o u l d e x p e c t
t h e e n d o m e t r i u r n to a c c u m u l a t e the s t e r o i d m o r e e f f e c t i v e l y than myometrium.
The p r e s e n t e x p e r i m e n t s u p p o r t s this
h y p o t h e s i s w h i c h m a y e x p l a i n the g r e a t e r s e n s i t i v i t y of the e n d o m e t r i u m to c h a n g e s in t h e l e v e l s of c i r c u l a t i n g e s t r o gens(12).
ACKNOW L E D G E M E N T S * T h i s i n v e s t i g a t i o n w a s a i d e d b y G r a n t #E307 f r o m t h e A m e r i c a n C a n c e r Society, and I n s t / t u t i o n a l G r a n t s f r o m the A m e r i c a n C a n c e r S o c i e t y (In 78} a n d U. S. P . H. S.
742
S T E R O I D S
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REFERENCES I. Flesher, J. W., Gupta, G. N., ffacobson, H. I. and Jensen, E. V. Fed. Proc. 19___2,170 (1960). Z. Jensen, E. V., and Jacobson, H. I. Recent Prog. H o r m o n e Res. 18_2 387 (1962). 3. Glascock, R. F. and Hoekstra, W. G. Biochem. Journal 72, 673, (1959). 4. Stone, G. M., Baggett, B. and Dormelly, R. B. J. Endocrin. 27, 271 (1963). 5. Stone, G. M . J. Endocrin. ZT, Z81 (1963). 6. De Paepe, J. C. Nature 185, 264 (1960). 7. Kalberer, F. and Rutschman, J. Helv. Chim. Act. 4_44, 1956 (1961). 8. Astwood, E. B. A m . J. Physiol. 126, 16Z (1939). 9. Talwar, O. P., Segal, S. J., Evans, H. and Davidson, O. W. Proc. Nat. Acad. Sci. 50, 1059 (1964). I0. Fishman, W . H., "Chemistry of Drug Metabolism". C. C. T h o m a s (1961) p 134. II. Fishman, W . H. and Baker, J. R. J. Histochem. and Cytochem. 4, 570 (1956). lZ. Allen, W . M . Anat. Rec. 48__, 65 (1931). 13. The abbreviations used in the text are as follows: P P O , Z, 5diphenyloxazole; P O P O P , p-bis (Z-(5-phenyloxazolyl))-benzene
PREFERENTIAL
UPTAKE
O F 6, 7 - H 3 - E S T R A D I O L B Y T H E
ENDOMETRIUM
OF THE RAT UTERUS~
/ames
W. F l e s h e r
D e p a r t m e n t of P h a r m a c o l o g y U n i v e r s i t y of K e n t u c k y Lexington, Kentucky
Received February
i0, 1965
ABSTRACT
T h e u p t a k e of t r i t i a t e d e s t r a d i o l b y the e n d o m e t r i u m a n d m y o m e t r i u m of the r a t u t e r u s h a s b e e n s t u d i e d f o l l o w i n g i n t r a venous administration. These data demonstrate the p r e f e r e n t i a l u p t a k e of r a d i o a c t i v i t y b y t h e e n d o m e t r i u m . Preferential u p t a k e b y the e n d o m e t r i u m m a y e x p l a i n the g r e a t e r s e n s i t i v i t y of t h e e n d o r n e t r i u r n to c h a n g e s in the l e v e l s of c i r c u l a t i n g e s t r o gens.
Previous
studies have shown that uterus and vagina are able
to t a k e up a n d r e t a i n m o r e of t r i t i a t e d e s t r a d i o l t h a n o t h e r t i s s u e s of t h e i m m a t u r e
r a t f o l l o w i n g s y s t e m i c a d m i n i s t r a t i o n ( i p Z).
Ear-
l l e r , i t w a s s h o w n t h a t h e x e s t r o l of h i g h s p e c i f i c a c t i v i t y r e a c h e s a maximum
l e v e l in the u t e r u s of i m m a t u r e
hours after injection(3).
More recently,
goats and sheep 2-6
a similar incorporation
p a t t e r n h a s b e e n f o u n d in the o v a r i e c t o m i z e d
mouse following
intravenous and subcutaneous administration
of t r i t l a t e d e s t r a d i o l
a n d e s t r o n e ( 4, 5).
Using autoradiography,
p h y s i o l o g i c a l a m o u n t s of t r i t i a t e d e s t r a d i o l
mice given hyperw e r e f o u n d to
738
S T E R O I D S
5:5
a c c u m u l a t e r a d i o a c t i v i t y in the u t e r i n e e n d o m e t r i u m ( 6 ) . T h e p r e s e n t p a p e r r e p o r t s on the u p t a k e of t r i t l a t e d e s t r a d i o l by t h e u t e r i n e e n d o m e t r l u r n a n d m y o m e t r i u r n of the y o u n g a d u l t rat~ w h e n a d m i n i s t e r e d in a p h y s i o l o g i c a l d o s e .
M ~ T E R . I A L S AND M E T H O D S
S o l u t i o n s : 6, 7 - H 3 - e s t r a d i o l - 1 7 B ( s p e c i f i c a c t i v i t y 3 6 . 8 c u r i e s / r n l i L i m o l e ) was p u r c h a s e d f r o m New E n g l a n d N u c l e a r C o r p , It w a s p u r i f i e d by d e s c e n d i n g p a p e r c h r o m a t o g r a p h y in a m o d i f i e d B u s h B - 3 s y s t e m : H e p t a n e 333 rnl, b e n z e n e I()7 m l , m e t h a n o l 400 rnl, w a t e r I00 m l . C o n t r o l s p o t s of n o n - r a d i o a c t i v e s t e r o i d w e r e d e t e c t e d on p a p e r c h r o m a t o g r a m s a f t e r d i p p i n g the c h r o m a t o g r a m s in a m i x t u r e of I% F e C I 3 a n d I% K F e (CN)6 a n d t h e w a s h i n g w i t h 6NHCI a n d f i n a l l y w i t h w a t e r . T h e z o n e c o r r e s p o n d i n g to c o n t r o l e s t r a d i o l w a s e l u t e d w i t h m e t h a n o l . T h e p u r i f i e d m a t e r i a l w a s e v a p o r a t e d w i t h N2, w i t h g e n t l e w a r m i n g , to a s m a l l v o l u m e a n d d i l u t e d w i t h b e n z e n e . R ~ d i o c h e r n i c a l p u r i t y was checked by a n a l y t l c a l p a p e r c h r o m a t o g r a p h y as follows: A f t e r d e v e l o p m e n t w i t h t h e s o l v e n t , ( B u s h B - 3 s y s t e m ) the c h r o r n a t o g r a m w a s d r i e d in a i r , c u t i n t o 1 c m s t r i p s a n d e l u t e d i n d i v l d u a l l y in c o u n t i n g b o t t l e s w i t h 5 m l of s p e c t r o g r a d e d i o x a n e . A f t e r s t a n d i n g o v e r n i g h t , I0 rnl of P p O ( 1 3 ) - t o l u e n e (4 g / l i t e r ) w a s a d d e d a n d t h e s a m p l e s w i t h the s t r i p s s t i l l in p l a c e w e r e c o u n t e d in t h e T r i - C a r b Liquid S c i n t i l l a t i o n S p e c t r o m e t e r . M o r e t h a n 96 p e r c e n t of t h e r a d i o a c t i v i t y w h i c h o c c u r r e d in a s i n g l e p e a k w a s f o u n d to be 6, 7 - H 3 - e s t r a d i o l - 1 7 B . P o r t i o n s of the m e t h a n o l - b e n z e n e s o l u t i o n w e r e e v a p o r a t e d w i t h N 2 to n e a r d r y n e s s . A b s o l u t e e t h a n o l w a s t h e n a d d e d a n d e v a p o r a t e d to n e a r d r y n e s s . T h e p r o c e d u r e w a s r e p e a t e d a n d the f i n a l s a l i n e s o l u t i o n ( 0 . 9 % N a C I ; I% e t h a n o l ) w a s m a d e up to c o n t a i n t h e s t e r o i d in a c o n c e n t r a t i o n of 0, 4 ~ g / m l , s p e c i f i c a c t i v i t y 100~c/ug" A n i m a l s : F e m a l e S p r a g u e - D a w l e y r a t s , 2 0 5 - 2 2 4 g, w e r e Lightly a n e s t h e t i z e d w i t h e t h e r a n d t h e s a p h e n o u s v e i n e x p o s e d f o r i n t r a v e n o u s i n j e c t i o n . T h e a n i m a l s w e r e s a c r i f i c e d by d e c a p i t a t i o n two h o u r s a f t e r t h e i n t r a v e n o u s a d m i n i s t r a t i o n of 0. l~g of b, 7 - H 3 - e s t r a d i o l p e r I00 g b o d y w e i g h t , u t e r i q u i c k l y removed and dissected from surrounding tissue. Each uterine horn was opened with a longitudinal cut and
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l a i d on a g l a s s s l i d e . T h e e n d o m e t r z u m t h u s e x p o s e d w a s s c r a p e d off w i t h t h e e d g e of a s e c o n d g l a s s s l i d e . H i s t o l o g i c a l e x a m i n a t i o n ( t i s s u e e m b e d d e d in p a r a f f i n , s e c t i o n s s t a i n e d with picric acid, acid fuchsin and hematoxylin) indicated that the p r o c e d u r e r e m o v e d a b o u t 75% of the e n d o m e t r i u r n . T h e e n d o m e t r i a l s a m p l e c o n t a i n e d no m y o m e t r i a l e l e m e n t s . I m m e d i a t e l y f o l l o w i n g s e p a r a t i o n of the t i s s u e s , t h e y w e r e t r a n s f e r r e d to t a r e d w e i g h i n g b o t t l e s . A f t e r w e i g h i n g , the s a m p l e w a s d r i e d a t h i g h v a c u u m a n d r e w e i g h e d . R a d i o A s s a y : C o m b u s t i o n s of d r i e d e n d o m e t r i u m , m y o m e t r i u r n a n d b l o o d w e r e c a r r i e d o u t by the o x y g e n - f l a s k t e c h n i q u e u s i n g a m o d i f i c a t i o n - of the m e t h o d of K a l b e r e r a n d R u t s c h m a n (7). S p e c t r o g r a d e d i o x a n e (11. O ml) w a s t r a n s f e r r e d i n t o the w e U of 1 l i t e r c o m b u s t i o n f l a s k . T h e d i o x a n e w a s f r o z e n by a c e t o n e - d r y - i c e m i x t u r e a n d the f l a s k c h a r g e d w i t h pure oxygen. Combustion was started with a projection lamp ( S y l v a n i a , T r u - F l e c t o r , 150 w a t t s ) . T h e d i o x a n e w a s m e l t e d by i m m e r s i n g the w e l l in w a r m w a t e r a n d the f l a s k r o t a t e d e n d o v e r e n d to i n s u r e c o m p l e t e m i x i n g a s p r e v i o u s l y d e s c r i b e d (7). A 10 m l p o r t i o n w a s w i t h d r a w n a n d a d d e d to 5 m l of phosphor consisting of 7.5 g P P O , 75 m g P O P O P , iZ0 g napthalene in 500 m l of xylene. Radioactivity was m e a s u r e d in a T r i - C a r b l i q u i d S c i n t i l l a t i o n S p e c t r o m e t e r w i t h a c o u n t i n g e f f i c i e n c y of Z5%.
RESULTS
AND
DISCUSSION
A s s h o w n in T a b l e I, two h o u r s a f t e r the i n t r a v e n o u s a d m i n i s t r a t i o n of 6, 7 - H 3 - e s t r a d l o l ,
t h e c o n c e n t r a t i o n of
r a d i o a c t i v i t y in e n d o m e t r i u r n is a b o u t two a n d o n e - h a l f t i m e s the c o n c e n t r a t i o n in m y o m e t r i u m .
A t the s a m e t i m e , the
l e v e l of r a d i o a c t i v i t y in b l o o d i s o n l y a p p r o x i m a t e l y 5-10% of the l e v e l in u t e r u s . T h e s e d a t a d e m o n s t r a t e the s e l e c t i v i t y of t h e e n d o m e t r i u m in i t s a b i l i t y to a c c u m t t l a t e r a d i o a c t i v i t y .
A l t h o u g h no a t t e m p t
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Table I
C o m p a r i s o n of l i q u i d - s c i n t i l l a t i o n c o u n t i n g of e n d o m e t r i u m a n d m y o m e t r i u r n o x i d i z e d by the o x y g e n - f l a s k t e c h n i q u e . Tissue
Relative Specific Activity
Endometriurn Myometrium RSA
=
% Wate r
228 + 16 92 + 12 counts/min/mg counts/min/mg
78-80 77-82
tissue whole uterus
x
100
V a l u e s e x p r e s s e d a s r e l a t i v e s p e c i f i c a c t i v i t i e s (RSA) a r e the m e a n + s t a n d a r d e r r o r f o r 6 d e t e r m i n a t i o n s .
w a s m a d e in t h e s e e x p e r i m e n t s to i d e n t i f y the r a d i o a c t i v i t y , i t h a s b e e n p r e v i o u s l y s h o w n t h a t o n l y e s t r a d i o l c o u l d be i s o l a t e d f r o m the i m m a t u r e
r a t u t e r u s ( 1, Z).
It i s t h e r e f o r e r e a s o n a b l e to a s s u m e the r a d i o a c t i v i t y o b s e r v e d is due to 6, 7 - H S - e s t r a d i o l - 1 7 B .
T h e f i n d i n g of
d i f f e r e n t c o n c e n t r a t i o n s of r a d i o a c t i v i t y in e n d o m e t r i u m s u g g e s t several possibilities which could account for these results; (I) a g r e a t e r n u m b e r of r e c e p t o r b i n d i n g s i t e s in e n d o m e t r i u r n than myometrium;
(Z) a m e c h a n i s m f o r the a c c u m u l a t i o n of
h o r m o n e in t a r g e t t i s s u e s ; (3) a h i g h w a t e r c o n t e n t in e n d o metrium,
w h i c h on d r y i n g the t i s s u e r e s u l t s in a h i g h s p e c i f i c
activity.
T h e t h i r d p o s s i b i l i t y w a s e x c l u d e d by t h e f i n d i n g t h a t the
w a t e r c o n t e n t i s the s a m e in e n d o m e t r i u m a n d m y o m e t r i u m ( T a b l e I).
A c c o r d i n g to A s t w o o d (8) t h e p e r c e n t a g e of w a t e r in
M a y 1965
S T E R O ID S
741
w h o l e u t e r u s d u r i n g t h e e s t r o u s c y c l e r a n g e s f r o m 7 9 . 8 to 84.8.
E v i d e n c e f o r t h e p r e s e n c e of r e c e p t o r s h a s b e e n p r e -
s e n t e d in s t u d i e s on t h e s u b c e U u l a r l o c a l i z a t i o n of 6, 7 - H 3e s t r a d i o 1 - 1 7 B in t h e u t e r u s of the o v a r i e c t o m / z e d r a t (9). hal/the
About
r a d i o a c t i v i t y w a s f o u n d a s s o c i a t e d w i t h t h e 105, 000 g
supernatant fraction, which apparently contains a specific "receptor"
substance(s) for estradlol.
s u c h a s l u n g do n o t b i n d e s t r a d i o l ,
Non-target tissues
evidently b e c a u s e the
a p p r o p r i a t e r n a c r o m o l e c u l e s a r e n o t p r e s e n t in l u n g t i s s u e . I t h a s b e e n p o s t u l a t e d by F i s h m a n t h a t t h e g l u c o s i d u r o n i c a c i d of e s t r a d i o l i s f o r m e d in t h e l i v e r a n d i n t e s t i n e a n d i s c a r r i e d to t h e " t a r g e t o r g a n " w h e r e B - g l u c u r o n i d a s e t r a n s f e r s t h e g l u c u r o n y 1 g r o u p to a s u i t a b l e a c c e p t o r a n d r e l e a s e s estradiol(10).
Evidence has also been presented that B-glucur-
o n i d a s e is p r e s e n t i n - h i g h e r c o n c e n t r a t i o n in e n d o m e t r i u m t h a n in m y o m e t r i u m ( 1 1 ) .
A c c o r d i n g to t h i s m o d e l , one w o u l d e x p e c t
t h e e n d o m e t r i u r n to a c c u m u l a t e the s t e r o i d m o r e e f f e c t i v e l y than myometrium.
The p r e s e n t e x p e r i m e n t s u p p o r t s this
h y p o t h e s i s w h i c h m a y e x p l a i n the g r e a t e r s e n s i t i v i t y of the e n d o m e t r i u m to c h a n g e s in t h e l e v e l s of c i r c u l a t i n g e s t r o gens(12).
ACKNOW L E D G E M E N T S * T h i s i n v e s t i g a t i o n w a s a i d e d b y G r a n t #E307 f r o m t h e A m e r i c a n C a n c e r Society, and I n s t / t u t i o n a l G r a n t s f r o m the A m e r i c a n C a n c e r S o c i e t y (In 78} a n d U. S. P . H. S.
742
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REFERENCES I. Flesher, J. W., Gupta, G. N., ffacobson, H. I. and Jensen, E. V. Fed. Proc. 19___2,170 (1960). Z. Jensen, E. V., and Jacobson, H. I. Recent Prog. H o r m o n e Res. 18_2 387 (1962). 3. Glascock, R. F. and Hoekstra, W. G. Biochem. Journal 72, 673, (1959). 4. Stone, G. M., Baggett, B. and Dormelly, R. B. J. Endocrin. 27, 271 (1963). 5. Stone, G. M . J. Endocrin. ZT, Z81 (1963). 6. De Paepe, J. C. Nature 185, 264 (1960). 7. Kalberer, F. and Rutschman, J. Helv. Chim. Act. 4_44, 1956 (1961). 8. Astwood, E. B. A m . J. Physiol. 126, 16Z (1939). 9. Talwar, O. P., Segal, S. J., Evans, H. and Davidson, O. W. Proc. Nat. Acad. Sci. 50, 1059 (1964). I0. Fishman, W . H., "Chemistry of Drug Metabolism". C. C. T h o m a s (1961) p 134. II. Fishman, W . H. and Baker, J. R. J. Histochem. and Cytochem. 4, 570 (1956). lZ. Allen, W . M . Anat. Rec. 48__, 65 (1931). 13. The abbreviations used in the text are as follows: P P O , Z, 5diphenyloxazole; P O P O P , p-bis (Z-(5-phenyloxazolyl))-benzene