Immunological demonstration of α-fetoprotein in uterine cytosol from immature rats

BIOCHIMIE, 1974, 56, 567-570.

Immunological demonstration of -fetoprotein in uterine cytosol from immature rats. Claude AUSSEL, Jos6

URIEL, Georges MICHEL ( ' ) a n d E t i e n n e - E m i l e BAULIEU ( ' ) .

Institut de Recherches Scientifiques sur le Cancer du C.N.R.S. 9 4 8 0 0 - V i l l e j u i f (*) Unitd de Recherches sur le M~tabolisme Mol~culaire et la Physio-Pathologie des St~ro~'des de H.N.S.E.R.M., H6pital de Bicdtre, 94270-Bicdtre. (10-12-1973). Summary. - - The identity between serum aFP and the estrogen binding 4-5 S macromolecular complex of uterine cytosol from immature rats has been demonstrated by the use of a specific immunoadsorbent to aFP. Analysis of the sedimentation profile in glycerol gradients of uterine cytosol incubated with tritiated estrone or estradiol suggests that the total estrogen binding capacity of the 4-5 S complex is provided by aFP. Changes of aFP content in rat uterus with the age of the animals indicate that this protein is probably present in the cytosol as a serum contaminant. a-fetoprotein (aFP) is the first a-globulin to appear i n m a m m a l i a n s e r u m d u r i n g development and the d o m i n a n t s e r u m p r o t e i n i n early emb r y o n i c life. The estrogen b i n d i n g activity of rat, mouse a n d h u m a n aFP has been recently demonstrated b y i m m u n o l o g i c a l methods [1]. Isolated rat a F P has a molecular weight of 72,000 daltons, a s e d i m e n t a t i o n coefficient of 4.5. S and b i n d s estrone a n d estradiol w i t h affinity c o n s t a n t of 1.17 X 10SM -1 a n d 5 X 107M -1, respectively [2], confirming the values obtained directly i n p l a s m a samples [3, 4]. The d y n a m i c s of synthesis a n d secretion of rat aFP d u r i n g ontogenic development a n d early postnatal life as studied by i m m u n o l o g i c a l method [5, 6] has s h o w n that serum c o n c e n t r a t i o n s as high as 2-4 m g / m l at b i r t h fall to several h u n d r e d m i c r o g r a m s b e t w e e n 21 a n d 25 days and to only a few h u n d r e d n a n o g r a m s at about 5 weeks. Traces (20'-30 n g / m l ) of a F P still r e m a i n p r e s e n t i n the s e r u m of adult a n i m a l s [7].

specific antisera a n d i m m u n o a d s o r b e n t s to rat aFP. The results o b t a i n e d suggest strongly that the estrogen b i n d i n g associated w i t h the so-called 4-5 p e g is due to aFP, w h i c h is p r e s e n t in u t e r i n e homogenate p r o b a b l y as a s e r u m c o n t a m i n a n t . MATERIAL AND METHODS. 6,7(3H)-estradiol (48 Ci/mmol) was o b t a i n e d from New E n g l a n d Nuclear, 6,7(all) - estrone (55 C i / m m o l ) from C.E.A. a n d purified by partition c o l u m n c h r o m a t o g r a p h y . Non-radioactive steroids w e r e o b t a i n e d from Roussel-UCLAF a n d tested for p u r i t y by t h i n layer c h r o m a t o g r a p h y .

It is w.ell established that homogenates from uterii of i m m a t u r e rats (21-25 days old) c o n t a i n macromoIecules called receptors w h i c h b i n d estrone a n d estradiol w i t h high affinity [8, 9] comp a r a b l e to rat aFP [2]. U l t r a c e n t r i f u g a t i o n sedim e n t a t i o n p a t t e r n s of radioactive estradiol a n d estrone b o u n d w i t h high affinity to m a c r o m o l e c u l e c o m p o n e n t s show peaks at about 8 S a n d 4-5 S a c c o r d i n g to the e x p e r i m e n t a l c o n d i t i o n s [15].

Specific antisera to rat a F P a n d to adult rat s e r u m p r o t e i n were o b t a i n e d as p r e v i o u s l y described [10]. Sheep a n t i b o d i e s to rat a F P w e r e isolated by a d s o r p t i o n a n d elution from i n s o h b i l i z e d rat a m n i o t i c fluid, using the p r o c e d u r e of Avrameas a n d T e r n y n c k [11]. Antibodies to adult rat s e r u m p r o t e i n s were isolated by the same p r o c e d u r e after a d s o r p t i o n a n d elution from i n s o l u b i l i z e d adult rat s e r u m proteins. Glycine-HC1 buffer 0.2 M pH 2.8 was used i n the elution steps. After neutralisation w i t h 1 M phosphate buffer pH 7.4 a n d dialysis against phosphate buffered saline, pH 7.4, the isolated a n t i b o d i e s were separatively coupled to glutaraldehyde-activated Biogel P 3 0 0 beads [12]. Both i m m u n o a d s o r b e n t s were e q u i l i b r a t e d before use w i t h Tris HC1 buffer (Tris 10 mM EDTA 1.5 raM) pH 7.4.

I n the p r e s e n t work, the possibility that the estrogen b i n d i n g component(s) of u t e r i n e cytosol from i m m a t u r e rats are, at least i n part, due to the presence of s e r u m aFP, was explored by the use of

The i m m u n o l o g i c a l q u a n t i t a t i o n of aFP was c a r r i e d out using the method of Macini [13]. A s t a n d a r d curve i n the range of 0:5 to 500 ~g of antigen w a s established w i t h a F P isolated b y the

C. A u s s e l , J. Uriel, G. M i c h e l et E . - E . B a u l i e u .

568

m e t h o d p r e v i o u s l y d e s c r i b e d [2]. P r o t e i n s w e r e d e t e r m i n e d by the m e t h o d of L o w r y [14] using Bovine Serum A l b u m i n as standard. I n n n a t u r e female W i s t a r rats (6, 8, 10 and 21 days) w e r e studied. After d e c a p i t a t i o n , the uterii w e r e q u i c k l y r e m o v e d , w a s h e d a n d h o m o genized at 0°C in 4-5 v o l u m e s of Tris HC1 buffer (Tris 10 mM, E D T A 1.5 mM, m e r c a p t o e t h a n o I 2 rnM, pH 7.4) w i t h the use of a teflon-glass h o m o genizer. Cytosol w a s p r e p a r e d by c e n t r i f u g a t i o n of the h o m o g e n a t e at 0°C and 105,000 g f o r 90 minutes. T h e cytosol w a s d i v i d e d in 2 aliquots. One was k e p t at 0°C and s e r v e d as the control. T h e other w a s t r e a t e d w i t h 1 ml of i m m u u o a d s o r b e n t anti-rat (~FP f o r 2 h at 0°C w i t h m i l d stirring. After c e n t r i f u g a t i o n (5,000 r p m 1.0 rain.), the supernatant w a s decanted. T h e i m m u n o a d s o r b e n t was w a s h e d and s u s p e n d e d in 2 ml buffer and r e c e n t r i fuged. This o p e r a t i o n was r e p e a t e d three times. T h e four s u p e r n a n t a n t s w e r e p o o l e d and c o n c e n t r a t e d to t h e i r initial volume, u n d e r ~¢acuum dialysis using S c h l e i s c h e r - S c h u l l ultrathimbles. Samples of c o n t r o l and i m m u n o a d s o r b e d cytosols w e r e i n c u b a t e d at 0°C for 90 rain. w i t h the d e s i r e d conc e n t r a t i o n of r a d i o a c t i v e steroids. Aliquots of 0.25 ml of the i n c u b a t e d cytosols w e r e c e n t r i f u g e d at 0°C t h r o u g h a l i n e a r 5-35 p. cent g l y c e r o l gradient. The a p p a r e n t s e d i m e n t a t i o n coefficients w e r e calculated using b o v i n e s e r u m a l b u m i n as refer e n c e standard. T w o drop f r a c t i o n s w e r e collected f r o m the bottom of the tubes and m i x e d w i t h 3 ml ethanol and 10 ml of t o l u e n e based scintillator. Counts w e r e c o r r e c t e d to 100 p. cent efficiency by e x t e r n a l s t a n d a r d i z a t i o n .

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R E S U L T S AND DISCUSSION.

S e r u m contamination of uterine homogenates. Uterii f r o m a group of 10 days old rats w e r e w a s h e d w i t h m i l d agitation at .0°C in t h r e e successive baths, 15 min. each, of I5 ml of n e u t r a l buffered saline and 2 X 15 ml of Tris-HC1 10 mM EDTA 1.5 mM buffer, pH 7.4. After the last step a cytosol e x t r a c t f r o m the w a s h e d uterii w a s preTABLE L

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BIOCHIMIE, 1974, 56, n ° 4.

p a r e d a c c o r d i n g t h e p r o c e d u r e given in ¢ Material and Methods ~. a F P w a s quantified by r a d i a l imm u n o d i f f u s i o n in the w a s h i n g solutions as well as in cytosol. Table I s u m m a r i z e s the results obtained. T h e slow decrease in the quantities of a F P lost as the w a s h i n g progresses suggests that t h e p r o t e i n m a y be p r e s e n t for the most p a r t in the e x t r a c e l l u l a r c o m p a r t m e n t . On the other h a n d , the high level of a F P r e m a i n i n g in the cytosol after the last w a s h i n g (18 ~ g / m l ) is c o m p a t i b l e w i t h its significant c o n t r i b u t i o n to the estrogen b i n d i n g c a p a c i t y of the p r e p a r a t i o n .

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b e t w e e n 6 a n d 21 days w e r e studied. Uterii f r o m each group of rats (10 to 32 p e r group) w e r e w a s h e d as i n d i c a t e d above, p o o l e d a n d h o m o g e n i zed (see <>). Sera w e r e also c o l l e c t e d and p o o l e d in the same m a n n e r . Content af a F P w a s d e t e r m i n e d in both s e r u m a n d cytosol f r o m different groups. The results are s h o w n in figure 1. S e r u m a F P p r o g r e s s i v e l y decreases w i t h age except, as r e p o r t e d p r e v i o u s l y [5], a r o u n d the l l t h day w h e r e a t r a n s i t o r y i n c r e a s e was observed. In cytosol, the decrease of u F P w i t h age r o u g h l y p a r a l l e l s s e r u m changes. This sugegsts that most, if not all, of a F P p r e s e n t in cytosol m a y be due to s e r u m c o n t a m i n a t i o n of u t e r i n e tissue.

I d e n t i t y of e F P and the uterine 4-5 S estrogen binding component. Rat u t e r i n e cytosol was i n c u b a t e d in the p r e s e n c e of i n s o l u b i l i z e d antibodies to eFP. T h e a n t i b o d y i m m u n o a d s o r b e n t w a s

569

a - f e t o p r o t e i n in u t e r i n e cgtosol. removed by c e n t r i f u g a t i o n and the eytosol supern a t a n t e q u i l i b r a t e d w i t h 10 nM 3H-estrone and subjected to glycerol g r a d i e n t sedimentation. It is k n o w n that labelling w i t h radioactive estradiol w o u l d have given a m a j o r 8 S pe~k a n d a relatively modest 4-5 S b i n d i n g [15]. Conversely estrone, in the intact cytosol, has relatively little affinity for the 8 S c o m p a r e d to estradiol, but b i n d s very m u c h in the 4-'5 S region. F i g u r e 2 shows the sedimentation profiles of 3H-estrone labelled control and a F P - i m m u n o a d s o r b e d u t e r i n e cytosols from 6 day old rats. It is a p p a r e n t that the large peak of

r a d i o a c t i v i t y peaks (fig. 3, A). The latter, but not the 8 S peak w h i c h r e m a i n e d u n c h a n g e d , disapeared largely after a F P i m m u n o a d s o r p t i o n . Quantitation of (tFP after glycerol g r a d i e n t s e d i m e n t a t i o n 10* 3 D P M / m g

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Fraction Number Fit;. 2. - - Density gradient sedimentation pattern of radioactivity in uterine cgtosol prepared from 6 day old rats. 0.25 ml of control cytosol ( e - - e ) or imnmnoadsorbed cytosol ( © - - © ) incubated with 10 nM of 3H-estrone were layered on a 5-35 p. cent linear glycerol gradient in Tris-EDTA. C-entrifugatipn was for 15 h. at 49,000 rpm in a Spinco SW 50.1 rotor.

r a d i o a c t i v i t y c o r r e s p o n d i n g to a s e d i m e n t a t i o n coefficient of 4-5 S p r e s e n t in control eytosol, completely disappears w h e n aFP is specifically removed from the u t e r i n e homogenate. Other e x p e r i m e n t s using 3H-estradiol 10 nM a n d 10 day old a n i m a l s have essentially confirmed the p r e v i o u s findings. The s e d i m e n t a t i o n profile of control cytosol showed the two 8 S and 4-5 S BIOCHIMIE, 1974, 56, n ° 4.

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Fit;. 3. - - A) Density gradient sedimentation pattern of radioactivity in uterine cytosol prepared from 10 day old rat. 0.25 ml control cytosol ( e - - e ) and immunoadsorbed eytosol ( O - - O ) were incubated with 10 nM 8H-estradiol. Centrifugation was carried as fig. 2. B) Distribution of e~FP in uterine cytosol fractionated by glycerol gradient centrifugation as in (A). (tFP was quantified by radial immunodiffusion.

of control cytosol showed a single peak of a F P in the 4-5 S zone (3, B). Conversely, no ctFP could be detected in a n y fraction of the glycerol g r a d i e n t of a F P - i m m u n o a d s o r b e d cytosol. No change was observed in the s e d i m e n t a t i o n profile of cytosol samples from either 6 day or 10 day old a n i m a l s after i n c u b a t i o n with an i m m u n o a d s o r b e n t to adult rat s e r u m proteins. F i n a l l y it seems 1) that the estrogen b i n d i n g activity associated w i t h the so-called 4-5 S u t e r i n e p r o t e i n s is p r o v i d e d by aFP i n the a n i m a l s studied, and 2) that this p r o t e i n is p r e s e n t i n u t e r i n e homogenate as a s e r u m c o n t a m i n e n t r a t h e r t h a n as an i n t r a e y t o p l a s m i e constituant. The f u n c t i o n a l r e l a t i o n s h i p s between the 8 S receptor and a F P r e m a i n u n e x p l a i n e d . F u r t h e r

C. A u s s e l , J. Uriel, G. M i c h e l et E . - E . B a u l i e u .

570

w o r k is n e c e s s a r y to e l u c i d a t e t h i s p r o b l e m a n d t h e r e f o r e t h e c o n t r i b u t i o n o f a F P to t h e r e s p o n s i v e n e s s of u t e r u s to e s t r o g e n s . R~suM~. L'utilisation d ' i m m u n o a d s o r h a n t s sp6cifiques antiu-foetoprot6ine a p e r m i s de d6montrer l'identit6 entre l'a-foetoprot6ine et le complexe macromol6culaire 4-5 S liant l'cestradiol dans le cytosol ut6rin de rates i m m a tures. L'analyse de profils obtenus apr~s centrifugation de eytosols, incub6s avec de l'cestrone on de l'oestradiol triti6, sur gradiant de glyc6rol suggbre que la eapacit6 de liaison d'mstrog6nes au complexe 4-5 S est due u n i q u e m e n t h 1'(~FP. Les changemeuis de concentration de I'uFP dans l'ut6rus de rate en fonction de l'fige indique que cette prot6ine est p r o b a b l e m e n t pr6sente dans le cytosol en qualit6 de c o n t a m i n a t i o n s6rique. BIBLIOGRAPHIE. 1. Uriel, J., de Nechaud, B. a Dupiers, M. (1972) Biochem. Biophys. Res. Commun., 46, 1175-1180. 2. Aussel, C., Uriel, J. ~ Mercier-Bodard, C. (1973) Biochimie, 55, 1431-1437.

BIOCHIMIE, 1974, 56, n ° 4.

3. Raynaud, J. P., Mercier-Bodard, C. ~ Baulieu, E. E. (1971) Steroids, 18, 767-788. 4. Nunez, E., EngeImann, F., Benassayag, C., Savu, L., Crepy, O. ~ Jayle, M. F. (1971) C. R. Acad. Sci. (Paris), 272, 2396-2399. 5. de Nechaud, B. ~ Uriel, J. (1971) Int. J. Cancer, 8, 71-80. 6. de Nechaud, B., Unpublished results. 7. Sell, S. & Gord, D, (1973) lmmunochemistry, 10, 439-444. 8. Best-Belpomme, M., Fries, J. ,~ Erdos, T. (1970) Eur. J. Biochem., 17, 425-430. 9. Truong, H. & Baulieu, E. E. (1971) Biochim. Biophys. Acta, 237, 167-175. 10. Michel, G., Jung, I., Baulieu, E. E., Aussel, C. & Uriel, J. Steroids (in press). 11. Stanislawski-Birencwajg, M. (1967) Cancer Res., 27, 1982-1989. 12. Avrameas, S. ~ Ternynck, T. (1969) Immunochemistry, 6, 53-66: 13. Ternynck, T. ~ Avrameas, S. (1972) FEBS Letters, 23, 24-28. 14. Mancini, G., Vaerman, S. P., Carbonara, A. D. & Heremans, J. F. (1964) Proc. Biol. Fluids, 12, 37O-375. 15. Lowry, O. M., Rosebrough, N. J., Farr, A. L. & Randall, J. (1951) J. Biol. Chem., 193, 265-268.