RAPID C OMMUNI C A T IO N
ZIPGRAM
EPIDERMAL GROWTH FACTOR-LIKE MATERIAL IN RAT SUBMANDIBULAR GLAND (1 ) EDWARD W. GRESIK, HENDRIKA VAN DER NIOEN AND TIBOR BARKA Department of Anatomy, Mount Sinai School of Medicine of the City University of New York, New York 10029
ABSTRACT By using an antiserum s p e c i f i c f o r mouse epidermal growth f a c t o r (EGF) , only t h e granular convoluted tubule (GCT) c e l l s revealed immunochemical s t a i n i n g i n r a t submandibular glands. There was no regular sexual difference i n t h e frequency o r s i z e of immunoreactive c e l l s . Extracts of gland contained an antigen w h i c h showed a complete crossr e a c t i v i t y w i t h mouse EGF i n radioimmunoassays. The r e l a t i v e amounts of EGF, determined by a heterologous radioimmunoassay, were not s i g n i f i cantly d i f f e r e n t i n t h e glands of r a t s of t h e two sexes. Administration of testosterone caused an increase, i n both sexes, in the number of GCT c e l l s stained f o r EGF and i n t h e amount of EGF i n t h e gland. There was no s i g n i f i c a n t sexual difference i n these two parameters a f t e r androgen treatment.
The submandibular glands of t h e r a t and mouse a r e s i m i l a r i n s t r u c t u r e (Tupa, '26).
However, a pronounced sexual dimorphism of the gland i s seen
only i n t h e mouse (Lacassagne, '40), but not i n t h e r a t (Cutler and Chaudhry, '75). Several biologically a c t i v e polypeptides have been localized i n considerable q u a n t i t i e s i n t h e granular convoluted tubule (GCT) segment of mouse submandibular gland (Junqueira e t a l .
, '49; Goldstein and Burdman, '65;
Bhoola e t a l . , ' 7 3 ; Gresik and Barka, ' 7 7 ) .
Some of these substances have
a l s o been detected i n f a r l e s s e r amounts i n t h e submandibular glands of r a t s (Riekkinen and Niemi, '68; de Jong e t a l .
, '72; Simson e t a l . , '78).
In the present study we provide evidence f o r the presence i n r a t submandibular gland of a f a c t o r which immunologically cross-reacts w i t h antiserum against mouse epidermal growth f a c t o r (EGF).
83
MATERIALS AND METHODS Ad u l t male and female Sprague-Dawley r a t s were fa s te d f o r 24 h be fo re k i l l i n g .
Rats were i n j e c t e d subcutaneously w i t h
20 mg o f testosterone propionate i n 0.4 m l o f sesame o i l one week before killing.
Untreated r a t s served as c o n t r o l s .
One submandibular gland was
f i x e d i n Bouin's s o l u t i o n and s ta i n e d immunocytochemically u s i n g an a n t i serum against p u r i f i e d mouse EGF, as described p r e v i o u s l y (Gresik and Barka, The o t h e r gland was homogenized i n c o l d d i s t i l l e d water.
'77).
enate was c e n t r i f u g e d a t 4'
The homog-
C f o r 20 min a t 15,000 rpm. The r e l a t i v e
amounts o f EGF i n th e supernatants were determined by a
heterologous r a d i o -
immunoassay using mouse-EGF as reference standard, as r a d i o i o d i n a t e d t r a c e r , and as antigen t o r a i s e antibodies i n r a b b i t s , as d e t a i l e d elsewhere (Barka e t al.,
'78a, '78b).
Student's t - t e s t was used t o c a l c u l a t e s i g n i f i c a n c e
o f d if f erence. RESULTS Rat submandibular gland contained a f a c t o r o r f a c t o r s which cross-reacted w i t h t h e antiserum a g a i n s t p u r i f i e d mouse EGF.
Competitive
b i n d ing curves f o r p u r i f i e d mouse EGF, as a standard, and f o r an e x t r a c t o f r a t submandibular gland were n o t p a r a l l e l ( f i g . 1).
Th i s may i n d i c a t e
complete c r o s s - r e a c t i v i t y o f mouse EGF w i t h t h e p r o t e i n ( s ) i n t h e gland e x t r a c t ( c f . Hunter, ' 7 8 ), b u t t h e o t h e r explanation, i.e., o f i n t e r f e r i n g substances, can n o t be excluded.
t h e presence
The immunoreactive sub-
stance i n t h e r a t gland henceforth w i l l be r e f e r r e d t o as EGF. The r e l a t i v e amounts o f EGF, as measured b y a heterologous r a d i o immunoassay, were n o t s i g n i f i c a n t l y d i f f e r e n t i n t h e glands of male and female rat s.
A s i n g l e i n j e c t i o n o f te s to s t e r o n e increased EGF s i g n i f i c a n t l y
t o t h e same l e v e l i n b o th sexes ( t a b l e 1). Immunocytochemical techniques demonstrated t h a t EGF was confined t o t h e GCT c e l l s i n t h e glands o f b o th sexes ( f i g s . 2, 3).
84
There was considerable
TABLE 1 The e f f e c t of testosterone on the weight and r e l a t i v e amount of EGF i n submandi bular glands EGF
male Control Testosterone-treated a: b:
11.7 26.1
female
f &
8.1 4.3 9.9a 24.4
? &
2.2 8.7b
ma1 e
W t . of gland female
156 k 14 171 f 24
156 160
? ?
12 9
P
ZIPGRAM
EPIDERMAL GROWTH FACTOR-LIKE MATERIAL IN RAT SUBMANDIBULAR GLAND (1 ) EDWARD W. GRESIK, HENDRIKA VAN DER NIOEN AND TIBOR BARKA Department of Anatomy, Mount Sinai School of Medicine of the City University of New York, New York 10029
ABSTRACT By using an antiserum s p e c i f i c f o r mouse epidermal growth f a c t o r (EGF) , only t h e granular convoluted tubule (GCT) c e l l s revealed immunochemical s t a i n i n g i n r a t submandibular glands. There was no regular sexual difference i n t h e frequency o r s i z e of immunoreactive c e l l s . Extracts of gland contained an antigen w h i c h showed a complete crossr e a c t i v i t y w i t h mouse EGF i n radioimmunoassays. The r e l a t i v e amounts of EGF, determined by a heterologous radioimmunoassay, were not s i g n i f i cantly d i f f e r e n t i n t h e glands of r a t s of t h e two sexes. Administration of testosterone caused an increase, i n both sexes, in the number of GCT c e l l s stained f o r EGF and i n t h e amount of EGF i n t h e gland. There was no s i g n i f i c a n t sexual difference i n these two parameters a f t e r androgen treatment.
The submandibular glands of t h e r a t and mouse a r e s i m i l a r i n s t r u c t u r e (Tupa, '26).
However, a pronounced sexual dimorphism of the gland i s seen
only i n t h e mouse (Lacassagne, '40), but not i n t h e r a t (Cutler and Chaudhry, '75). Several biologically a c t i v e polypeptides have been localized i n considerable q u a n t i t i e s i n t h e granular convoluted tubule (GCT) segment of mouse submandibular gland (Junqueira e t a l .
, '49; Goldstein and Burdman, '65;
Bhoola e t a l . , ' 7 3 ; Gresik and Barka, ' 7 7 ) .
Some of these substances have
a l s o been detected i n f a r l e s s e r amounts i n t h e submandibular glands of r a t s (Riekkinen and Niemi, '68; de Jong e t a l .
, '72; Simson e t a l . , '78).
In the present study we provide evidence f o r the presence i n r a t submandibular gland of a f a c t o r which immunologically cross-reacts w i t h antiserum against mouse epidermal growth f a c t o r (EGF).
83
MATERIALS AND METHODS Ad u l t male and female Sprague-Dawley r a t s were fa s te d f o r 24 h be fo re k i l l i n g .
Rats were i n j e c t e d subcutaneously w i t h
20 mg o f testosterone propionate i n 0.4 m l o f sesame o i l one week before killing.
Untreated r a t s served as c o n t r o l s .
One submandibular gland was
f i x e d i n Bouin's s o l u t i o n and s ta i n e d immunocytochemically u s i n g an a n t i serum against p u r i f i e d mouse EGF, as described p r e v i o u s l y (Gresik and Barka, The o t h e r gland was homogenized i n c o l d d i s t i l l e d water.
'77).
enate was c e n t r i f u g e d a t 4'
The homog-
C f o r 20 min a t 15,000 rpm. The r e l a t i v e
amounts o f EGF i n th e supernatants were determined by a
heterologous r a d i o -
immunoassay using mouse-EGF as reference standard, as r a d i o i o d i n a t e d t r a c e r , and as antigen t o r a i s e antibodies i n r a b b i t s , as d e t a i l e d elsewhere (Barka e t al.,
'78a, '78b).
Student's t - t e s t was used t o c a l c u l a t e s i g n i f i c a n c e
o f d if f erence. RESULTS Rat submandibular gland contained a f a c t o r o r f a c t o r s which cross-reacted w i t h t h e antiserum a g a i n s t p u r i f i e d mouse EGF.
Competitive
b i n d ing curves f o r p u r i f i e d mouse EGF, as a standard, and f o r an e x t r a c t o f r a t submandibular gland were n o t p a r a l l e l ( f i g . 1).
Th i s may i n d i c a t e
complete c r o s s - r e a c t i v i t y o f mouse EGF w i t h t h e p r o t e i n ( s ) i n t h e gland e x t r a c t ( c f . Hunter, ' 7 8 ), b u t t h e o t h e r explanation, i.e., o f i n t e r f e r i n g substances, can n o t be excluded.
t h e presence
The immunoreactive sub-
stance i n t h e r a t gland henceforth w i l l be r e f e r r e d t o as EGF. The r e l a t i v e amounts o f EGF, as measured b y a heterologous r a d i o immunoassay, were n o t s i g n i f i c a n t l y d i f f e r e n t i n t h e glands of male and female rat s.
A s i n g l e i n j e c t i o n o f te s to s t e r o n e increased EGF s i g n i f i c a n t l y
t o t h e same l e v e l i n b o th sexes ( t a b l e 1). Immunocytochemical techniques demonstrated t h a t EGF was confined t o t h e GCT c e l l s i n t h e glands o f b o th sexes ( f i g s . 2, 3).
84
There was considerable
TABLE 1 The e f f e c t of testosterone on the weight and r e l a t i v e amount of EGF i n submandi bular glands EGF
male Control Testosterone-treated a: b:
11.7 26.1
female
f &
8.1 4.3 9.9a 24.4
? &
2.2 8.7b
ma1 e
W t . of gland female
156 k 14 171 f 24
156 160
? ?
12 9
P
