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Insulin-like immunoreactivity in cells of the mouse anterior pituitary
Micron, 1981, V o l . : 12, p p . 2 0 5 - 2 0 6 . ©Pergamon P r e s s Ltd. P r i n t e d i n G r e a t B r i t a i n .
0047-7206/81/020205-02502.00/0
INSULIN-LIKE IMMUNOREACTIVITY IN CELLS OF THE MOUSE ANTERIOR PITUITARY
James S. H a t f i e l d , Ben Pansky 1, H a r d r e s s J . W a l l e r 2 ' 5 , and G. C o l i n Budd 3 D e p a r t m e n t o f Anatomy, Wayne S t a t e U n i v e r s i t y School o f M e d i c i n e , D e t r o i t , M i c h i g a n 48201 and D e p a r t m e n t s o f Anatomyl~ N e u r o s c i e n c e s 2, and P h y s i o l o g y 5, Medical C o l l e g e o f Ohio, C.S. 10008, T o l e d o , Ohio 43699
I n s u l i n - l i k e i m m u n o r e a c t i v i t y h a s r e c e n t l y been o b s e r v e d i n c e l l s o f v a r i o u s b r a i n r e g i o n s , i n c l u d i n g t h e h y p o t h a l a m u s and median eminence, w i t h t h e use o f b o t h i n d i r e c t i m m u n o f l u o r e s c e n c e and immunoperoxidase t e c h n i q u e s ( 1 , 2 ) . Because o f t h e c l o s e a n a t o m i c and p h y s i o l o g i c r e l a t i o n s h i p o f t h e p i t u i t a r y t o t h e s e b r a i n r e g i o n s , we have b e e n l e d t o examine t h e p i t u i t a r y f o r endogenous i n s u l i n - l i k e i m m u n o r e a c t i v i t y w i t h an i n d i r e c t immunoperoxidase t e c h n i q u e adapted for electron microscopy. Mature NZB mice were p e r f u s e d w i t h b u f f e r e d s a l i n e f o l l o w e d by 1.25% g l u t a r a l d e h y d e . The p i t u i t a r i e s were removed, f i x e d o v e r n i g h t i n 1.25% g l u t a r a l d e hyde, and r o u t i n e l y p r o c e s s e d and embedded i n Epon, w i t h t h e o m i s s i o n o f o s m i c a t i o n . Thin s e c t i o n s o f t h e a n t e r i o r p i t u i t a r y mounted on n i c k e l g r i d s were e t c h e d w i t h 5% H202 f o r 10 m i n u t e s , and i n c u b a t e d w i t h 5% normal s h e e p serum f o r one h o u r t o r e d u c e n o n - s p e c i f i c immunoreactivity. The s e c t i o n s were t h e n i n c u b a t e d f o r 24 t o 48 h o u r s w i t h g u i n e a p i g a n t i - p o r c i n e i n s u l i n , d i l u t e d 1 : i 0 0 , f o l l o w e d by g o a t a n t i - g u i n e a p i t IgG, d i l u t e d 1 : 5 0 , f o r one h o u r . The s e c t i o n s were r e a c t e d w i t h H a n k e r - Y a t e s r e a g e n t s ( p h e n y l e n e d i a m i n e / p y r o c a t e c h o l ) f o r 5 m i n u t e s , o s m i c a t e d w i t h 4% OsO4, and l i g h t l y s t a i n e d w i t h u r a n y l a c e t a t e . The s e c t i o n s were r i n s e d t h r e e t i m e s f o r f i v e m i n u t e s i n 0 . 1 M T r i s b u f f e r , pH 7 . 6 , b e t w e e n e a c h s t e p o f t h e procedure. Controls for antibody specificity included absorption of the first antiserum with p o r c i n e i n s u l i n , s e l e c t i v e removal o f i n s u l i n a n t i b o d i e s w i t h a S e p h a r o s e column, and o m i s sion of the first or second antiserum. C o n t r o l s e c t i o n s e x h i b i t e d no s p e c i f i c a c c u m u l a t i o n s of reaction product (Fig. 1). S e c t i o n s i n c u b a t e d w i t h b o t h a n t i s e r a c o n t a i n e d c e l l s i n which i m m u n o r e a c t i o n p r o d u c t was p r o m i n e n t l y d e p o s i t e d on s e c r e t o r y g r a n u l e s . Such g r a n u l e s were most o f t e n p e r i p h e r a l l y d i s t r i b u t e d a l o n g t h e i n s i d e o f t h e c e l l membrane, and e x t e n d i n g linearly along the processes of the cells (Fig. 2). C e l l s which c o n t a i n e d i m m u n o r e a c t i o n p r o d u c t were c h a r a c t e r i s t i c a l l y angular in appearance, with processes often extending to the b a s e m e n t membrane o f a d j a c e n t b l o o d v e s s e l s ( F i g . 3 ) . The s e c r e t o r y g r a n u l e s , a p p r o x i m a t e l y 100 t o 200 nm i n s i z e , were c l e a r l y d e l i n e a t e d by t h e e l e c t r o n - d e n s e r e a c t i o n p r o d u c t , i n c o n t r a s t t o u n r e a c t i v e s e c r e t o r y g r a n u l e s i n n e a r b y c e l l s o f d i f f e r e n t morphology ( F i g . 4 ) . The s i z e o f t h e i m m u n o r e a c t i v e s e c r e t o r y g r a n u l e s , t h e a n g u l a r i t y o f t h e c e l l s , and t h e p r o c e s s e s c o n t a i n i n g l i n e a r l y a r r a y e d g r a n u l e s and e x t e n d i n g t o w a r d s b l o o d v e s s e l s a r e c h a r a c t e r i s t i c o f c e l l s i d e n t i f i e d as c o r t i c o t r o p h s and t h y r o t r o p h s ( 5 ) . These o b s e r v a t i o n s do n o t i n d i c a t e w h e t h e r t h e p r e s e n c e o£ i n s u l i n ( o r an i n s u l i n - l i k e s u b s t a n c e ) i s t h e r e s u l t o f de novo s y n t h e s i s by t h e s e c e l l s , o r u p t a k e o f t h e s u b s t a n c e from t h e v a s c u l a t u r e . However, t h e presence of insulin-like immunoreactivity in a specific cell type of the anterior pituitary strongly suggests a role for insulin in the regulation of pituitary functions.
(1)
Pansky, B. and H a t f i e l d , J . S . , 1978. C e r e b r a l l o c a l i z a t i o n o f i n s u l i n by i m m u n o f l u o r escence. Am. J . A n a t . , 153: 459-467. (2) " H a t f i e l d , J.S~-~,Pansky, B., W a l l e r , H . J . , and Budd, G.C., 1979. I m m u n o p e r o x i d a s e - l a b e l ed i n s u l i n a n t i b o d y l o c a l i z a t i o n i n mouse h y p o t h a l a m u s and p i t u i t a r y . J. Cell Biol. 83: 138a. (3) Nakane, P.K., 1970. C l a s s i f i c a t i o n s o f a n t e r i o r p i t u i t a r y c e l l s w i t h immnoenzyme histochemistry. J. Histo. ~ C~tochem., 18: 9-20.
S u p p o r t e d i n p a r t by B i o m e d i c a l R e s e a r c h S u p p o r t G r a n t No. SO 7 RR 05700-09 and by a g r a n t from t h e H e a l t h F o u n d a t i o n , U n i t e d Way o f C e n t r a l S t a r k County. 205
206
J.S. Hatfield, B. Pansky, H.J. Waller and G.C. Budd
Figures 1-4: Transmission electron micrographs of the mouse anterior pituitary. I. A control section shows no specific immunoreactivity upon elimination of the primary antiserum. Other-controls were similar in appearance, x 7800 2. Several angular cells exhibit peripherally arrayed secretory granules which are intensi f ~ d by the electron-dense i~unoreaction product. In contrast, the larger, unreactlve granules in an adjacent cell are pale (arrowheads). x5200 3. A cell containing immunoreactive secretory granules abutts the basement membrane (arrowheads) of an adjacent blood vessel (by). x6300 4. Electron-dense secretory granules are visible in a process extending between two unreactive cells. Although some background reactivity is evident, the larger secretory granules in the adjacent cells are unreactive (arrowheads). x23,000
0047-7206/81/020205-02502.00/0
INSULIN-LIKE IMMUNOREACTIVITY IN CELLS OF THE MOUSE ANTERIOR PITUITARY
James S. H a t f i e l d , Ben Pansky 1, H a r d r e s s J . W a l l e r 2 ' 5 , and G. C o l i n Budd 3 D e p a r t m e n t o f Anatomy, Wayne S t a t e U n i v e r s i t y School o f M e d i c i n e , D e t r o i t , M i c h i g a n 48201 and D e p a r t m e n t s o f Anatomyl~ N e u r o s c i e n c e s 2, and P h y s i o l o g y 5, Medical C o l l e g e o f Ohio, C.S. 10008, T o l e d o , Ohio 43699
I n s u l i n - l i k e i m m u n o r e a c t i v i t y h a s r e c e n t l y been o b s e r v e d i n c e l l s o f v a r i o u s b r a i n r e g i o n s , i n c l u d i n g t h e h y p o t h a l a m u s and median eminence, w i t h t h e use o f b o t h i n d i r e c t i m m u n o f l u o r e s c e n c e and immunoperoxidase t e c h n i q u e s ( 1 , 2 ) . Because o f t h e c l o s e a n a t o m i c and p h y s i o l o g i c r e l a t i o n s h i p o f t h e p i t u i t a r y t o t h e s e b r a i n r e g i o n s , we have b e e n l e d t o examine t h e p i t u i t a r y f o r endogenous i n s u l i n - l i k e i m m u n o r e a c t i v i t y w i t h an i n d i r e c t immunoperoxidase t e c h n i q u e adapted for electron microscopy. Mature NZB mice were p e r f u s e d w i t h b u f f e r e d s a l i n e f o l l o w e d by 1.25% g l u t a r a l d e h y d e . The p i t u i t a r i e s were removed, f i x e d o v e r n i g h t i n 1.25% g l u t a r a l d e hyde, and r o u t i n e l y p r o c e s s e d and embedded i n Epon, w i t h t h e o m i s s i o n o f o s m i c a t i o n . Thin s e c t i o n s o f t h e a n t e r i o r p i t u i t a r y mounted on n i c k e l g r i d s were e t c h e d w i t h 5% H202 f o r 10 m i n u t e s , and i n c u b a t e d w i t h 5% normal s h e e p serum f o r one h o u r t o r e d u c e n o n - s p e c i f i c immunoreactivity. The s e c t i o n s were t h e n i n c u b a t e d f o r 24 t o 48 h o u r s w i t h g u i n e a p i g a n t i - p o r c i n e i n s u l i n , d i l u t e d 1 : i 0 0 , f o l l o w e d by g o a t a n t i - g u i n e a p i t IgG, d i l u t e d 1 : 5 0 , f o r one h o u r . The s e c t i o n s were r e a c t e d w i t h H a n k e r - Y a t e s r e a g e n t s ( p h e n y l e n e d i a m i n e / p y r o c a t e c h o l ) f o r 5 m i n u t e s , o s m i c a t e d w i t h 4% OsO4, and l i g h t l y s t a i n e d w i t h u r a n y l a c e t a t e . The s e c t i o n s were r i n s e d t h r e e t i m e s f o r f i v e m i n u t e s i n 0 . 1 M T r i s b u f f e r , pH 7 . 6 , b e t w e e n e a c h s t e p o f t h e procedure. Controls for antibody specificity included absorption of the first antiserum with p o r c i n e i n s u l i n , s e l e c t i v e removal o f i n s u l i n a n t i b o d i e s w i t h a S e p h a r o s e column, and o m i s sion of the first or second antiserum. C o n t r o l s e c t i o n s e x h i b i t e d no s p e c i f i c a c c u m u l a t i o n s of reaction product (Fig. 1). S e c t i o n s i n c u b a t e d w i t h b o t h a n t i s e r a c o n t a i n e d c e l l s i n which i m m u n o r e a c t i o n p r o d u c t was p r o m i n e n t l y d e p o s i t e d on s e c r e t o r y g r a n u l e s . Such g r a n u l e s were most o f t e n p e r i p h e r a l l y d i s t r i b u t e d a l o n g t h e i n s i d e o f t h e c e l l membrane, and e x t e n d i n g linearly along the processes of the cells (Fig. 2). C e l l s which c o n t a i n e d i m m u n o r e a c t i o n p r o d u c t were c h a r a c t e r i s t i c a l l y angular in appearance, with processes often extending to the b a s e m e n t membrane o f a d j a c e n t b l o o d v e s s e l s ( F i g . 3 ) . The s e c r e t o r y g r a n u l e s , a p p r o x i m a t e l y 100 t o 200 nm i n s i z e , were c l e a r l y d e l i n e a t e d by t h e e l e c t r o n - d e n s e r e a c t i o n p r o d u c t , i n c o n t r a s t t o u n r e a c t i v e s e c r e t o r y g r a n u l e s i n n e a r b y c e l l s o f d i f f e r e n t morphology ( F i g . 4 ) . The s i z e o f t h e i m m u n o r e a c t i v e s e c r e t o r y g r a n u l e s , t h e a n g u l a r i t y o f t h e c e l l s , and t h e p r o c e s s e s c o n t a i n i n g l i n e a r l y a r r a y e d g r a n u l e s and e x t e n d i n g t o w a r d s b l o o d v e s s e l s a r e c h a r a c t e r i s t i c o f c e l l s i d e n t i f i e d as c o r t i c o t r o p h s and t h y r o t r o p h s ( 5 ) . These o b s e r v a t i o n s do n o t i n d i c a t e w h e t h e r t h e p r e s e n c e o£ i n s u l i n ( o r an i n s u l i n - l i k e s u b s t a n c e ) i s t h e r e s u l t o f de novo s y n t h e s i s by t h e s e c e l l s , o r u p t a k e o f t h e s u b s t a n c e from t h e v a s c u l a t u r e . However, t h e presence of insulin-like immunoreactivity in a specific cell type of the anterior pituitary strongly suggests a role for insulin in the regulation of pituitary functions.
(1)
Pansky, B. and H a t f i e l d , J . S . , 1978. C e r e b r a l l o c a l i z a t i o n o f i n s u l i n by i m m u n o f l u o r escence. Am. J . A n a t . , 153: 459-467. (2) " H a t f i e l d , J.S~-~,Pansky, B., W a l l e r , H . J . , and Budd, G.C., 1979. I m m u n o p e r o x i d a s e - l a b e l ed i n s u l i n a n t i b o d y l o c a l i z a t i o n i n mouse h y p o t h a l a m u s and p i t u i t a r y . J. Cell Biol. 83: 138a. (3) Nakane, P.K., 1970. C l a s s i f i c a t i o n s o f a n t e r i o r p i t u i t a r y c e l l s w i t h immnoenzyme histochemistry. J. Histo. ~ C~tochem., 18: 9-20.
S u p p o r t e d i n p a r t by B i o m e d i c a l R e s e a r c h S u p p o r t G r a n t No. SO 7 RR 05700-09 and by a g r a n t from t h e H e a l t h F o u n d a t i o n , U n i t e d Way o f C e n t r a l S t a r k County. 205
206
J.S. Hatfield, B. Pansky, H.J. Waller and G.C. Budd
Figures 1-4: Transmission electron micrographs of the mouse anterior pituitary. I. A control section shows no specific immunoreactivity upon elimination of the primary antiserum. Other-controls were similar in appearance, x 7800 2. Several angular cells exhibit peripherally arrayed secretory granules which are intensi f ~ d by the electron-dense i~unoreaction product. In contrast, the larger, unreactlve granules in an adjacent cell are pale (arrowheads). x5200 3. A cell containing immunoreactive secretory granules abutts the basement membrane (arrowheads) of an adjacent blood vessel (by). x6300 4. Electron-dense secretory granules are visible in a process extending between two unreactive cells. Although some background reactivity is evident, the larger secretory granules in the adjacent cells are unreactive (arrowheads). x23,000