ZIPGRAM
RAPID COMMUNICATION
TANNIC A C I D AS AN ELECTRON-DENSE PROBE I N THE TESTIS (1) AGUSTIN A O K I ( 2 ) , J I L L E I N S T E I N AND DON W. FAWCETT Department o f Anatomy, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT The use o f t a n n i c a c i d t o preserve and promote t h e s t a i n i n g o f p r o t e i n c o n s t i t u e n t s o f t h e e x t r a c e l l u l a r f l u i d i s des c r i b e d . I t s usefulness f o r d e l i n e a t i n g t h e e x t r a c e l l u l a r compartment and d e t e c t i n g changes i n c a p i l l a r y p e r m e a b i l i t y i s i l l u s t r a t e d i n experiments on t h e t e s t i s . The use o f t a n n i c a c i d (TA) as an a d d i t i v e t o t h e f i x a t i v e f o r t i s s u e f i n e s t r u c t u r e was i n t r o d u c e d r e c e n t l y by M i z u h i r a and Futaesaku ( ' 7 2 ) .
They found
t h a t TA forms complexes w i t h p r o t e i n s / p o l y p e p t i d e s and heavy metals y i e l d i n g a p r e c i p i t a t e o f high electron density.
T h e o r e t i c a l l y , t h e small m o l e c u l a r w e i g h t
o f TA (about 1,700) makes i t a d e s i r a b l e t r a c e r w i t h which t o s t u d y t h e i n t e r faces o f a d j o i n i n g c e l l s . i n c e l l junctions.
It may prove e s p e c i a l l y u s e f u l f o r d e t e c t i n g gaps
I t was o f p a r t i c u l a r i n t e r e s t t o us t o t r y t h i s compound
i n the t e s t i s i n order t o gain f u r t h e r i n s i g h t i n t o the organization o f the complex o c c l u d i n g j u n c t i o n s o f S e r t o l i c e l l s t h a t c o n s t i t u t e t h e s t r u c t u r a l b a s i s o f t h e b l o o d - t e s t i s b a r r i e r (BTB) (Fawcett e t a l . ,
METHODS
'70).
Testes o f a n e s t h e t i z e d r a t s were exposed through an i n c i s i o n o f
t h e scrotum and t h e t e s t i c u l a r a r t e r y was cannulated w i t h a 25 gauge needle conn e c t e d t o a 140-cm p e r f u s i o n l i n e ( f i g . 1). The spermatic c o r d and pampiniform plexus were then s e c t i o n e d and t h e p e r f u s i o n s t a r t e d s i m u l t a n e o u s l y w i t h a s h o r t wash o f s a l i n e f o l l o w e d by 2% TA ( r e a g e n t grade, F i s h e r S c i e n t i f i c Co.) ( 3 ) and 4% g l u t a r a l d e h y d e i n 0.05
M phosphate b u f f e r , pH 7.2, f o r 15 minutes.
Small
b l o c k s were c u t from f i x e d t i s s u e s , washed 3 t i m e s i n s a l i n e , t r e a t e d w i t h 1% 449
osmium tetroxide in 0 . 1 M phosphate buffer and embedded in a r a l d i t e . RESULTS
Preliminary observations on TA-perfused t e s t i s revealed no d i f -
ferences in e i t h e r interstitiurn o r seminiferous tubules when compared with prope r controls fixed without TA.
I n one r a t , however, the i n f i l t r a t i o n of the in-
t e r s t i t i u m of a t e s t i s with TA, due t o an accidental puncture of the cannulated a r t e r y , resulted in strong staining of a component of the lymph and of the f l u i d bathing the Leydig c e l l s .
This component, presumably a protein o r proteins,
formed an electron-opaque complex with TA, e a s i l y visualized in the electron microscope a f t e r lead s t a i n i n g ( f i g . 2 ) .
This observation subsequently was con-
firmed in a s e r i e s of r a t s injected with TA d i r e c t l y into the t e s t i c u l a r i n t e r s t i tium.
The TA-protein complex constituted a useful probe delineating the
e x t r a c e l l u l a r space, which had a d i s t r i b u t i o n s i m i l a r t o t h a t found with other electron-opaque t r a c e r s t h a t have been used in the study of the blood-testis barrier.
The bulk of t h i s complex f i l l e d the lymphatic sinusoids.
However i t
a l s o i n f i l t r a t e d the a c e l l u l a r lamellae of the peritubular t i s s u e and penetrated the i n t e r c e l l u l a r c l e f t s around sperrnatogenic c e l l s , delineating the basal compartment of the seminiferous tubules.
A small amount was occasionally seen
permeating the i n i t i a l portions of the junctions between S e r t o l i c e l l s b u t
FIGURE LEGENDS 1 Sketch of a cannulation of the spermatic a r t e r y with a 25 gauge needle cemented t o polyethylene tubing. 2 Normal r a t t e s t i s injected i n t e r s t i t i a l l y w i t h tannic acid in glutaraldehyde. The peri tubul ar lymphatic sinusoids and i n t e r c e l l u l a r spaces of the boundary t i s s u e of the seminiferous tubules a r e f i l l e d with a heavily stained complex of tannic acid and proteins. 3 T e s t i s of an animal an hour a f t e r injection of cadmium. The tannic acid, which normally does not traverse the c a p i l l a r y wall when perfused, has leaked out of the damaged c a p i l l a r i e s i n abundance, f i l l ing the spaces between myoid c e l l s and penetrating i n t o the i n t e r c e l l u l a r c l e f t between S e r t o l i c e l l s ( a t arrows) , below the S e r t o l i j unc t i onal compl ex.
450
never extending beyond these s t r u c t u r e s . The f a i l u r e of the TA t o produce any staining in normal t e s t i s a f t e r f i x ation by perfusion could be a t t r i b u t e d t o e i t h e r (1) lack of bind substance or ( 2 ) impermeability of the t e s t i c u l a r vessels t o TA.
The f i r s t p o s s i b i l i t y can
obviously be discarded as shown by i n t e r s t i t i a l injection of TA.
On the other
hand, the f a i l u r e of TA t o traverse the c a p i l l a r y endothelium i s not surprising, inasmuch as i t s poor a b i l i t y t o permeate biological membranes has limited i t s use in electron microscopic cytochemistry (Futaesaku and Mizuhira, ' 7 4 ) . Tannic acid i s a pentadigalloyl compound of glucose and in s p i t e of i t s molecular weight, which i s f a r l e s s than t h a t of c o l l o i d s , i t has long been considered as a colloidal micelle, probably because the mu1 t i v a l e n t molecules can e a s i l y e s t a b l i s h H-bonds t o yield larger polymers.
This p o s s i b i l i t y was
tested in conditions of a1 tered capi 1 l a r y permeabi 1 i t y a f t e r a subcutaneous injection of cadmium chloride.
In the r a t , as well a s in many other rodents,
cadmium s a l t s cause an increase in the permeabil i t y o€ the t e s t i c u l a r vessels which r e s u l t s in subsequent atrophy of the t e s t i s (Aoki and Hoffer, unpublished observations). Between 1 and 2 hr. following the Cd i n j e c t i o n , there was a massive diffusion of TA into the interstitiurn ( f i g . 3 ) .
In the lymphatics, the
precipitated TA-protein complex appeared l e s s compact than a f t e r d i r e c t i n t e r s t i t i a l i n j e c t i o n , possibly because of the massive extravasation o f f l u i d and edema of the interstitiurn under these conditions. of TA was similar t o t h a t described above.
Otherwise, the distribution
The leakage of TA from t e s t i c u l a r
vessels in Cd-treated r a t s was detected e a r l i e r than with any other t r a c e r procedure used before, and can be used t o advantage in the study of the f i r s t component of the blood-testis b a r r i e r ( B T B ) .
The c a p i l l a r y endothelium of the
t e s t i s has been neglected as a physiological b a r r i e r because i t i s known t o be highly permeable t o a number of endogenous as well as exogenous compounds. 452
Nevertheless a l t e r a t i o n of i t s permeability seems
tG
play a key r o l e in patho-
logical changes occurring in Cd-treated r a t s which eventually lead t o the des t r u c t i o n of t h e t e s t i s .
Tannic acid a s a probe could a l s o be used t o t e s t the
e f f e c t s of other experimental treatments t h a t may modify c a p i l l a r y permeability. LITERATURE C I T E D Fawcett, D. W., L. V. Leak and P. M. Heidger. 1970 Electron microscopic observations on the s t r u c t u r a l components o f the bloodt e s t i s b a r r i e r . J . Reprod. Fert. (Suppl.) 10: 105-122. Futaesaku , K . , and V. Mizuhira. 1974 Fixation of mechanism of tann i c acid and d-catecin. Application 1: A detection of oxidase a c t i v i t y by the use of d-catecin. 8 t h I n t . Cong. Electron Microscopy. Canberra Vol. 11, pp. 142-143. Mizuhira, V., and Y. Futaesaku. 1972 New f i x a t i o n f o r biological membranes using tannic acid. Acta Histochem. Cytochem. , 5: 233-236. REFERENCES 1 Supported by Research Grant N . I . H,?HD-02344. 2 Present address: Departamento de Quimica Macromolecular, Escuela T6cnica Superior de Ingenieros I n d u s t r i a l e s , Barcelona 14, Spain. 3 Commercially available tannic acids a r e complex mixtures of polyphenol i c compounds with a gal 1 oyl ated glucose s t r u c t u r e . They vary considerably in molecular weights. The r e s u l t s reported here r e f e r to the one preparation. Tannic acid from other sources might we1 1 y i e l d somewhat d i f f e r e n t r e s u l t s .
453
RAPID COMMUNICATION
TANNIC A C I D AS AN ELECTRON-DENSE PROBE I N THE TESTIS (1) AGUSTIN A O K I ( 2 ) , J I L L E I N S T E I N AND DON W. FAWCETT Department o f Anatomy, Harvard Medical School, Boston, Massachusetts 02115 ABSTRACT The use o f t a n n i c a c i d t o preserve and promote t h e s t a i n i n g o f p r o t e i n c o n s t i t u e n t s o f t h e e x t r a c e l l u l a r f l u i d i s des c r i b e d . I t s usefulness f o r d e l i n e a t i n g t h e e x t r a c e l l u l a r compartment and d e t e c t i n g changes i n c a p i l l a r y p e r m e a b i l i t y i s i l l u s t r a t e d i n experiments on t h e t e s t i s . The use o f t a n n i c a c i d (TA) as an a d d i t i v e t o t h e f i x a t i v e f o r t i s s u e f i n e s t r u c t u r e was i n t r o d u c e d r e c e n t l y by M i z u h i r a and Futaesaku ( ' 7 2 ) .
They found
t h a t TA forms complexes w i t h p r o t e i n s / p o l y p e p t i d e s and heavy metals y i e l d i n g a p r e c i p i t a t e o f high electron density.
T h e o r e t i c a l l y , t h e small m o l e c u l a r w e i g h t
o f TA (about 1,700) makes i t a d e s i r a b l e t r a c e r w i t h which t o s t u d y t h e i n t e r faces o f a d j o i n i n g c e l l s . i n c e l l junctions.
It may prove e s p e c i a l l y u s e f u l f o r d e t e c t i n g gaps
I t was o f p a r t i c u l a r i n t e r e s t t o us t o t r y t h i s compound
i n the t e s t i s i n order t o gain f u r t h e r i n s i g h t i n t o the organization o f the complex o c c l u d i n g j u n c t i o n s o f S e r t o l i c e l l s t h a t c o n s t i t u t e t h e s t r u c t u r a l b a s i s o f t h e b l o o d - t e s t i s b a r r i e r (BTB) (Fawcett e t a l . ,
METHODS
'70).
Testes o f a n e s t h e t i z e d r a t s were exposed through an i n c i s i o n o f
t h e scrotum and t h e t e s t i c u l a r a r t e r y was cannulated w i t h a 25 gauge needle conn e c t e d t o a 140-cm p e r f u s i o n l i n e ( f i g . 1). The spermatic c o r d and pampiniform plexus were then s e c t i o n e d and t h e p e r f u s i o n s t a r t e d s i m u l t a n e o u s l y w i t h a s h o r t wash o f s a l i n e f o l l o w e d by 2% TA ( r e a g e n t grade, F i s h e r S c i e n t i f i c Co.) ( 3 ) and 4% g l u t a r a l d e h y d e i n 0.05
M phosphate b u f f e r , pH 7.2, f o r 15 minutes.
Small
b l o c k s were c u t from f i x e d t i s s u e s , washed 3 t i m e s i n s a l i n e , t r e a t e d w i t h 1% 449
osmium tetroxide in 0 . 1 M phosphate buffer and embedded in a r a l d i t e . RESULTS
Preliminary observations on TA-perfused t e s t i s revealed no d i f -
ferences in e i t h e r interstitiurn o r seminiferous tubules when compared with prope r controls fixed without TA.
I n one r a t , however, the i n f i l t r a t i o n of the in-
t e r s t i t i u m of a t e s t i s with TA, due t o an accidental puncture of the cannulated a r t e r y , resulted in strong staining of a component of the lymph and of the f l u i d bathing the Leydig c e l l s .
This component, presumably a protein o r proteins,
formed an electron-opaque complex with TA, e a s i l y visualized in the electron microscope a f t e r lead s t a i n i n g ( f i g . 2 ) .
This observation subsequently was con-
firmed in a s e r i e s of r a t s injected with TA d i r e c t l y into the t e s t i c u l a r i n t e r s t i tium.
The TA-protein complex constituted a useful probe delineating the
e x t r a c e l l u l a r space, which had a d i s t r i b u t i o n s i m i l a r t o t h a t found with other electron-opaque t r a c e r s t h a t have been used in the study of the blood-testis barrier.
The bulk of t h i s complex f i l l e d the lymphatic sinusoids.
However i t
a l s o i n f i l t r a t e d the a c e l l u l a r lamellae of the peritubular t i s s u e and penetrated the i n t e r c e l l u l a r c l e f t s around sperrnatogenic c e l l s , delineating the basal compartment of the seminiferous tubules.
A small amount was occasionally seen
permeating the i n i t i a l portions of the junctions between S e r t o l i c e l l s b u t
FIGURE LEGENDS 1 Sketch of a cannulation of the spermatic a r t e r y with a 25 gauge needle cemented t o polyethylene tubing. 2 Normal r a t t e s t i s injected i n t e r s t i t i a l l y w i t h tannic acid in glutaraldehyde. The peri tubul ar lymphatic sinusoids and i n t e r c e l l u l a r spaces of the boundary t i s s u e of the seminiferous tubules a r e f i l l e d with a heavily stained complex of tannic acid and proteins. 3 T e s t i s of an animal an hour a f t e r injection of cadmium. The tannic acid, which normally does not traverse the c a p i l l a r y wall when perfused, has leaked out of the damaged c a p i l l a r i e s i n abundance, f i l l ing the spaces between myoid c e l l s and penetrating i n t o the i n t e r c e l l u l a r c l e f t between S e r t o l i c e l l s ( a t arrows) , below the S e r t o l i j unc t i onal compl ex.
450
never extending beyond these s t r u c t u r e s . The f a i l u r e of the TA t o produce any staining in normal t e s t i s a f t e r f i x ation by perfusion could be a t t r i b u t e d t o e i t h e r (1) lack of bind substance or ( 2 ) impermeability of the t e s t i c u l a r vessels t o TA.
The f i r s t p o s s i b i l i t y can
obviously be discarded as shown by i n t e r s t i t i a l injection of TA.
On the other
hand, the f a i l u r e of TA t o traverse the c a p i l l a r y endothelium i s not surprising, inasmuch as i t s poor a b i l i t y t o permeate biological membranes has limited i t s use in electron microscopic cytochemistry (Futaesaku and Mizuhira, ' 7 4 ) . Tannic acid i s a pentadigalloyl compound of glucose and in s p i t e of i t s molecular weight, which i s f a r l e s s than t h a t of c o l l o i d s , i t has long been considered as a colloidal micelle, probably because the mu1 t i v a l e n t molecules can e a s i l y e s t a b l i s h H-bonds t o yield larger polymers.
This p o s s i b i l i t y was
tested in conditions of a1 tered capi 1 l a r y permeabi 1 i t y a f t e r a subcutaneous injection of cadmium chloride.
In the r a t , as well a s in many other rodents,
cadmium s a l t s cause an increase in the permeabil i t y o€ the t e s t i c u l a r vessels which r e s u l t s in subsequent atrophy of the t e s t i s (Aoki and Hoffer, unpublished observations). Between 1 and 2 hr. following the Cd i n j e c t i o n , there was a massive diffusion of TA into the interstitiurn ( f i g . 3 ) .
In the lymphatics, the
precipitated TA-protein complex appeared l e s s compact than a f t e r d i r e c t i n t e r s t i t i a l i n j e c t i o n , possibly because of the massive extravasation o f f l u i d and edema of the interstitiurn under these conditions. of TA was similar t o t h a t described above.
Otherwise, the distribution
The leakage of TA from t e s t i c u l a r
vessels in Cd-treated r a t s was detected e a r l i e r than with any other t r a c e r procedure used before, and can be used t o advantage in the study of the f i r s t component of the blood-testis b a r r i e r ( B T B ) .
The c a p i l l a r y endothelium of the
t e s t i s has been neglected as a physiological b a r r i e r because i t i s known t o be highly permeable t o a number of endogenous as well as exogenous compounds. 452
Nevertheless a l t e r a t i o n of i t s permeability seems
tG
play a key r o l e in patho-
logical changes occurring in Cd-treated r a t s which eventually lead t o the des t r u c t i o n of t h e t e s t i s .
Tannic acid a s a probe could a l s o be used t o t e s t the
e f f e c t s of other experimental treatments t h a t may modify c a p i l l a r y permeability. LITERATURE C I T E D Fawcett, D. W., L. V. Leak and P. M. Heidger. 1970 Electron microscopic observations on the s t r u c t u r a l components o f the bloodt e s t i s b a r r i e r . J . Reprod. Fert. (Suppl.) 10: 105-122. Futaesaku , K . , and V. Mizuhira. 1974 Fixation of mechanism of tann i c acid and d-catecin. Application 1: A detection of oxidase a c t i v i t y by the use of d-catecin. 8 t h I n t . Cong. Electron Microscopy. Canberra Vol. 11, pp. 142-143. Mizuhira, V., and Y. Futaesaku. 1972 New f i x a t i o n f o r biological membranes using tannic acid. Acta Histochem. Cytochem. , 5: 233-236. REFERENCES 1 Supported by Research Grant N . I . H,?HD-02344. 2 Present address: Departamento de Quimica Macromolecular, Escuela T6cnica Superior de Ingenieros I n d u s t r i a l e s , Barcelona 14, Spain. 3 Commercially available tannic acids a r e complex mixtures of polyphenol i c compounds with a gal 1 oyl ated glucose s t r u c t u r e . They vary considerably in molecular weights. The r e s u l t s reported here r e f e r to the one preparation. Tannic acid from other sources might we1 1 y i e l d somewhat d i f f e r e n t r e s u l t s .
453
