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Preputial glands of the albino mouse. Still, Susan 1971

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THE PREPUTIAL GLANDS OF THE ALBINO MOUSE b y SUSAN STILL B. Sc., Dalhousie U n i v e r s i t y , 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Zoology We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1971 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e tha t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r ag ree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumb ia V a n c o u v e r 8, Canada ABSTRACT The p a i r e d p r e p u t i a l glands of the a l b i n o mouse, Mus musculus, (Swiss s t r a i n ) were s t u d i e d w i t h r e g a r d to h i s t o l o g y , p h y s i o l o g y and b a s i c h i s t o c h e m i s t r y . F o r the h i s -t o l o g i c a l s tudy of the development and subsequent a g i n g of the p r e p u t i a l g l a n d , the glands of male and female mice of d i f f e r e n t ages, r a n g i n g from newborn t o 12 months o l d , were used. I n o r d e r to study the e f f e c t s of androgens and e s t r o g e n s upon the macroscopic and m i c r o s c o p i c s t r u c t u r e of the p r e p u t i a l g l a n d , w h i t e Swiss mice were d i v i d e d i n t o f o u r major groups ( a c c o r d i n g t o age and s t a t u s ) each of which were, i n t u r n , d i v i d e d i n t o subgroups a, b, c_, and d f o r t r e a t m e n t ; mice i n subgroups a and b r e c e i v e d d a i l y subcutaneous i n j e c t i o n s of t e s t o s t e r o n e p r o p i o n a t e and of e s t r a d i o l benzoate r e s p e c t i v e l y ; mice i n subgroup £ r e c e i v e d d a i l y subcutaneous i n j e c t i o n s of the v e h i c l e , sesame o i l ; mice i n subgroup d s e r v e d as c o n t r o l s . There were, of c o u r s e , male and female mice i n each g r o u p i n g . Routine h i s t o c h e m i c a l t e s t s f o r l i p i d s and p r o t e i n s were c a r r i e d out on the e x c i s e d p r e p u t i a l glands of w e a n l i n g and of young a d u l t mice, of b o t h sexes, i n o r d e r t o l o c a l i z e and t o i d e n t i f y the s e c r e t o r y p r o d u c t s of the a c i n a r c e l l s . H i s t o l o g i c a l l y and p h y s i o l o g i c a l l y ( i n terms of response to androgens and e s t r o g e n s ) the p r e p u t i a l glands of the mouse are s i m i l a r , i n most r e s p e c t s , to those of the - i i i -r a t as d e s c r i b e d by o t h e r a u t h o r s . However, t h e r e are apparent d i f f e r e n c e s i n the m i c r o s c o p i c anatomy and i n the h i s t o c h e m i s t r y of these homologous organs; f i r s t l y , the presence of an i n t i m a t e a s s o c i a t i o n of the mouse p r e p u t i a l g l a n d w i t h a c e n t r a l h a i r f o l l i c l e w h ich p e r s i s t s throughout the l i f e t i m e of the animal and which i s not found i n the r a t g l a n d ; s e c o n d l y , the absence of p e r i n u c l e a r p r o t e i n a c e o u s g r a n u l e s i n the a c i n a r c e l l s of the mouse p r e p u t i a l g l a n d w hich are p r e s e n t i n the a c i n a r c e l l s of the r a t p r e p u t i a l g l a n d , i n a d d i t i o n t o l i p i d d r o p l e t s . L i p i d s , p r i m a r i l y i n the form of d r o p l e t s of n e u t r a l f a t s a r e , t o my knowledge, the o n l y s e c r e t o r y p r o d u c t s of the mouse p r e p u t i a l g l a n d . D e v e l o p m e n t a l l y , h i s t o l o g i c a l l y , and h i s t o c h e m i c a l l y , the p r e p u t i a l g l a n d of the mouse i s an e x c e s s i v e l y developed sebaceous g l a n d and u n l i k e the r a t p r e p u t i a l g l a n d which cannot be c a t e g o r i z e d so s i m p l y , belongs t o the "monocrine" v a r i e t y of h o l o c r i n e g l a n d . - i v -TABLE OF CONTENTS Page T i t l e Page . i A b s t r a c t i i Table of Contents i v L i s t of Tables v L i s t of Graphs v i L i s t of P l a t e s v i i I n t r o d u c t i o n 1 M a t e r i a l s and Methods 3 R e s u l t s 11 Gross Anatomy of the A d u l t P r e p u t i a l Gland .. 11 M i c r o s c o p i c Anatomy of the A d u l t Gland 16 Development and A g i n g . 20 P h y s i o l o g y 27 H i s t o c h e m i s t r y 48 D i s c u s s i o n 54 H i s t o l o g y 54 P h y s i o l o g y 59 H i s t o c h e m i s t r y 75 Summary and C o n c l u s i o n s 78 I l l u s t r a t i o n s 82 Acknowledgements 101 L i t e r a t u r e C i t e d 102 -V-LIST OF TABLES TABLE PAGE I Summary of Experimental Procedure f o r the P h y s i o l o g i c a l Study . 6 II Normal V a r i a t i o n s i n Body Weight and Weights of S i n g l e P r e p u t i a l Glands i n Weanling and Young Adu l t Mice 1 5 I I I Average R e l a t i v e Weights of S i n g l e P r e p u t i a l Glands i n the P h y s i o l o g i c a l Study 29 IV Computer Program A n a l y s i s of the P h y s i o l o g i c a l Study 32 - v i -LIST OF GRAPHS GRAPH PAGE 1 Body Weights.and Weights of S i n g l e P r e p u t i a l Glands of Male Mice From 1 Week to 12 Months 13 2 Body Weights and Weights of S i n g l e P r e p u t i a l Glands of Female Mice From 1 Week to 12 Months 14 - v i i -LIST OF PLATES PLATE PAGE 1 The Young A d u l t P r e p u t i a l Gland 82 2 The Young A d u l t P r e p u t i a l Gland 83 3 The Development of the P r e p u t i a l Gland .. 84 4 The Development of the P r e p u t i a l Gland . . 85 5 The Aging of the P r e p u t i a l Gland 86 6 The A g i n g of the P r e p u t i a l Gland 87 7 The E x p e r i m e n t a l Glands of Group I (Males) 88 .8 The E x p e r i m e n t a l Glands of Group I (Males) 89 9 The E x p e r i m e n t a l Glands of Group I ' (Females ) 90 10 The E x p e r i m e n t a l Glands of Group I (Females) 91 11 The E x p e r i m e n t a l Glands of Group I i -I n t a c t A d u l t Mice (Males) 92 12 The E x p e r i m e n t a l Glands of Group I i -I n t a c t A d u l t Mice (Females) 93 13 The E x p e r i m e n t a l Glands of Group I I -V e a n l i n g Mice (Males) 94 14 The E x p e r i m e n t a l Glands of Group I I -Weanling Mice (Females) 95 15 The E x p e r i m e n t a l Glands of Group I l l -Newborn Mice 96 16 The E x p e r i m e n t a l Glands of Group I l l -Newborn Mice 97 - v i i i -PLATE PAGE 17 H i s t o c h e m i s t r y 98 18 H i s t o c h e m i s t r y 99 19 H i s t o c h e m i s t r y 100 - 1 -INTRODUCTION The p r e p u t i a l g l and s of the mouse, Mus muscu l u s , are p a i r e d subcutaneous organs w h i c h a re s i t u a t e d , i n b o t h s e xe s , i n the g e n i t a l r e g i o n of the a n i m a l , one on e i t h e r s i d e of the m i d l i n e . Each g l a n d opens i n d e p e n d e n t l y onto the s k i n , b e s i d e the u r e t h r a l open i n g , v i a an e x c r e t o r y d u c t w h i c h runs a l o n g the l a t e r a l s i d e of the p e n i s i n the male mouse and a l o n g the l a t e r a l s i d e of the c l i t o r i s i n the female mouse. U n l i k e the r a t p r e p u t i a l g l and s w h i c h have been the conce rn of d e t a i l e d h i s t o l o g i c a l , h i s t o c h e m i c a l , e n d o c r i n o -l o g i c a l and b i o c h e m i c a l i n v e s t i g a t i o n s by a number of a u t h o r s , the p r e p u t i a l g l and s of the mouse have r e c e i v e d c o m p a r a t i v e l y l i t t l e a t t e n t i o n . A l t h o u g h Voss (1932) and S c h a f f e r (1933) g i v e b r i e f d e s c r i p t i o n s of the g l and s i n the normal a d u l t male mouse, t h e i r d e t a i l e d morpho logy , t o my knowledge, has not been d e s c r i b e d . Other r e f e r e n c e s t o the mouse p r e p u t i a l g l a n d c once rn c u r s o r y e n d o c r i n o l o g i c a l i n v e s t i g a t i o n s i n v o l v i n g the re sponse of the g l a n d to androgens (Vos s , 1932; B u r d i c k and Gamon, 1941) and more r e c e n t l y , i n f o r m a t i o n on a p o s s i b l e sex a t t r a c t a n t f u n c t i o n f o r the g l a n d (B ron son , 1966; Gaunt, 1967 ) . My s t udy r e p o r t s the o b s e r v a t i o n s made upon the h i s t o l o g y of the p r e p u t i a l g l ands of norma l mice d u r i n g p o s t - n a t a l deve lopment and a g i n g and a l s o the h i s t o l o g y of - 2 -the glands of e x p e r i m e n t a l a n i m a l s , a f t e r hormone t r e a t m e n t s , i n order to determine t h e i r p o s s i b l e e n d o c r i n e r e l a t i o n s h i p s . F i n a l l y , r o u t i n e h i s t o c h e m i c a l t e s t s f o r l i p i d s and p r o t e i n s were c a r r i e d out on e x c i s e d p r e p u t i a l g l a n d s i n o r d e r t o i d e n t i f y and to l o c a l i z e t h e i r s e c r e t o r y p r o d u c t s . The paper of Beaver ( i 9 6 0 ) on the p r e p u t i a l glands of the r a t proved a v a l u a b l e p o i n t of r e f e r e n c e f o r my s t u d y on the mouse p r e p u t i a l g l a n d . - 3 -MATERIALS AND METHODS H i s t o l o g y Fo r the h i s t o l o g i c a l s tudy of the deve lopment and subsequent a g i n g of the p r e p u t i a l g l a n d , w h i t e Swis s m i c e , b o t h male and f e m a l e , of d i f f e r e n t ages r a n g i n g f rom newborn t o 12 months o l d were s a c r i f i c e d and the p a i r e d g l and s removed. I n m i c e , f rom b i r t h u n t i l 7 days of age, i t was n e c e s s a r y to d i s s e c t the whole g e n i t a l a r e a . A n i m a l s were s a c r i f i c e d a t week l y i n t e r v a l s f rom 7 days of age u n t i l 8 weeks of age and t h e r e a f t e r a t month l y i n t e r v a l s u n t i l 12 months of age. The d i s s e c t e d g l and s were f i x e d i m m e d i a t e l y i n B o u i n ' s f i x a t i v e f o r a p p r o x i m a t e l y 24 h o u r s . W i t h the e x c e p t i o n of newborn, 1, 2, 3, 4 , 5, and 6 day o l d g l and s wh i ch c o u l d not be i d e n t i f i e d g r o s s l y and t h e r e f o r e c o u l d no t be we i ghed , a l l g l and s were t r e a t e d s i m i l a r l y a f t e r f i x a t i o n , p r i o r t o w e i g h i n g . The g l and s were d i s s e c t e d f r e e of exces s p e r i g l a n d u l a r f a t , b l o t t e d d r y , and t h e n we ighed on a S a r t o r i u s b a l a n c e . F i n a l l y , a l l the g l and s were embedded i n p a r a f f i n , s e c t i o n e d s e r i a l l y a t 8 p.. and s t a i n e d w i t h D e l a f i e l d ' s h e m a t o x y l i n and c o u n t e r s t a i n e d w i t h e o s i n . P h y s i o l o g y : Hormone Treatments F o r the p h y s i o l o g i c a l s t udy w h i t e mice of the Swis s • s t r a i n were d i v i d e d i n t o f o u r major groups a c c o r d i n g t o age and s t a t u s . Group I c o n s i s t e d of young a d u l t mice (age 7 weeks) wh i ch a t the onset of the expe r imen t were c a s t r a t e d _4-(by s u r g i c a l r emova l of the gonads t h r ough an abdomina l i n c i s i o n ) and had the l e f t p r e p u t i a l g l a n d removed a t the same t i m e . Fo r t h i s o p e r a t i o n the mice were a n a e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l i n j e c t i o n of A v e r t i n s o l u t i o n . (0.025 m l . of A v e r t i n pe r one gram of body w e i g h t was an adequate dosage. The A v e r t i n was p r e p a r e d f rom the f o l l o w i n g s t o c k s o l u t i o n : 1.0 m l . amylene h y d r a t e , 0.5 m l . t r i b r o m o -e t h a n o l , 1.0 m l . d i s t i l l e d w a t e r . Then 0.5 m l . of the s t o c k was made up t o 20 m l . of s o l u t i o n by the a d d i t i o n of 19.5 m l . of d i s t i l l e d w a t e r . ) Group I i c o n s i s t e d of young a d u l t mice (age 7 weeks) wh i ch a t the b e g i n n i n g of the exper iment had the l e f t p r e p u t i a l g l a n d removed under A v e r t i n a n a e s t h e s i a bu t were not c a s t r a t e d . Group I I was made up of w e a n l i n g mice (age 3 weeks) wh i ch had the l e f t p r e p u t i a l g l a n d removed a t the onset of the e x p e r i m e n t . F i n a l l y , Group I I I c o n s i s t e d of i n t a c t newborn m i c e . Each of t he se f o u r major groups was t h e n d i v i d e d i n t o subgroups a , b, £, and d a c c o r d i n g t o the t r e a t m e n t g i v e n . There we re , of c o u r s e , male and female mice i n each g r o u p i n g . Each mouse i n subgroup a r e c e i v e d a d a i l y subcutaneous i n j e c t i o n of 0.125 mg.of t e s t o s t e r o n e p r o p i o n a t e * i n 0.05 m l . of sesame o i l ( *Pe randren 25mg/cm , C i b a Company L i m i t e d ) . Each mouse i n subgroup b r e c e i v e d a d a i l y subcutaneous i n j e c t i o n of 0.00165 mg. of e s t r a d i o l b e n z o a t e * i n 0.05 m l . of sesame o i l ( ^ N u t r i t i o n a l B i o c h e m i c a l s C o r p o r a t i o n ) . I n subgroup c_ each a n i m a l was i n j e c t e d d a i l y w i t h 0.05 m l . of sesame o i l U.S.P. The mice i n subgroup d were not i n j e c t e d or h a n d l e d a f t e r the o r i g i n a l -5-operation, but served as controls for the other subgroups within the same major group. A l l groups I, I i , and III animals were kept on the experiment for 21 days. Group II animals, however, were on the experiment for 19 days only. Table I summarizes the above experimental procedure. At the end of each experiment,24 hours after the l a s t i n j e c t i o n , a l l the mice were s a c r i f i c e d and the remaining preputial gland, that i s the right gland, was removed from each animal i n groups I, I i , and II and both preputial glands were removed from each animal i n group I I I . As soon as the preputial glands were dissected, either at the onset of each experiment or at the end of each experiment, they were treated s i m i l a r l y . As previously described for the h i s t o l o g i c a l study, the glands were fixed i n Bouin's f i x a t i v e and thereafter dissected free of excess f a t , blotted dry, and weighed. (The paired glands from each mouse of group I I I , the newborn group, were weighed together and then averaged; the two glands from each animal of the adult and weanling groups (I, I i , II) were weighed singly so that the weights before and after treatment could be compared.) F i n a l l y , the glands were embedded i n paraf f i n , s e r i a l l y sectioned at 8 ji. and stained with hematoxylin and eosin. Histochemistry Routine histochemical tests for l i p i d s and proteins were carried out on the excised preputial glands of young ; adult Swiss mice (5-8 weeks) and of weanling mice, of both TABLE I SUMMARY OF EXPERIMENTAL PROCEDURE FOR THE PHYSIOLOGICAL STUDY Group Sex and D a i l y Treatment No. of Dosage/ Subgroup Animals Mouse T o t a l Hormone I n j e c t e d / Mouse D u r a t i o n Age and s t a t u s of a t onset of Experiment Experiment a ) T e s t o s t e r o n e M 5 0.125 mg 2. 625 mg 21 days Young a d u l t mice p r o p i o n a t e F 5 (age 7 "weeks) , c a s t r a t e d and b ) E s t r a d i o l M 5 0.00165 mg 0. 03465 mg 21 days l e f t p r e p u t i a l T benzoate F 3 g l a n d removed s i m u l t a n e o u s l y a t c)Sesame O i l M 5 0.05 ml 21 days b e g i n n i n g of U.S.P. F 5 experiment d ) C o n t r o l M F 2 4 21 days a ) T e s t o s t e r o n e M 3 0.125 mg 2. 625 mg 21 days I n t a c t , young p r o p i o n a t e F 3 a d u l t mice (age b ) E s t r a d i o l 7 weeks). L e f t M 3 0.00165 mg 0. 03465 mg 21 days p r e p u t i a l g l a n d benzoate F 2 removed a t I i b e g i n n i n g of c)Sesame O i l M 1 0.05 ml 21 days experiment U.S.P. F 1 d ) C o n t r o l M 7 21 days F 6 TABLE I cont. T o t a l Sex and Daily- Hormone D u r a t i o n Age and s t a t u s Treatment No. of Dosage/ I n j e c t e d / of a t onset of Group Subgroup Animals Mouse Mouse Experiment Experiment a ) T e s t o s t e r o n e M 3 0.125 mg 2.375 mg 19 days I n t a c t v e a n l i n g p r o p i o n a t e P 5 mice (age 3 weeks) b ) E s t r a d i o l L e f t p r e p u t i a l M 4 0.00165 mg 0.03135 mg 19 days g l a n d removed a t I I benzoate F 5 onset of experimen -c)Sesame O i l M 5 0.05 ml 19 days U.S.P. F 6 d ) C o n t r o l M F 5 6 19 days a ) T e s t o s t e r o n e M 4 0.125 mg 2.625 mg 21 days I n t a c t newborn I I I p r o p i o n a t e F 6 mice b ) E s t r a d i o l M 3 0.00165 mg 0.03465 mg 21 days benzoate F 4 c)Sesame O i l M 4 0.05 ml 21 days U.S.P. F 5 - 8 -s e x e s , i n o r de r t o l o c a l i z e and to i d e n t i f y , a t l e a s t , as t o c l a s s , the s e c r e t o r y p r o d u c t s of the a c i n a r c e l l s . A l l g l a nd s f o r h i s t o c h e m i c a l s t u d i e s were f i x e d i n 10%> b u f f e r e d n e u t r a l f o r m a l i n , u n l e s s , o t h e r w i s e , i n d i c a t e d by the method. The b u f f e r was sodium pho spha te . F i x a t i o n t ime v a r i e d f rom 12-24 hours depend ing upon the r e q u i r e m e n t of the p r o c e d u r e . The h i s t o c h e m i c a l and s t a i n i n g methods were c a r r i e d out a c c o r d i n g t o Pea r se (1968) excep t as o t h e r w i s e i n d i c a t e d . These methods i n c l u d e d McManus' Sudan b l a c k B t e c h n i q u e f o r compound l i p i d s i n p a r a f f i n s e c t i o n s ( a f t e r f i x a t i o n i n B a k e r ' s f o r m o l c a l c i u m f o r 4 w e e k s ) ; the p r o p y l e n e g l y c o l -Sudan b l a c k B method f o r l i p i d s ( P e a r s e , 1961) , u s i n g f r e s h f r o z e n s e c t i o n s of t i s s u e , about 14 ja i n t h i c k n e s s ; the Sudan b l a c k B method of Chayen, B i t e n s k y , B u t c h e r , and P o u l t e r ( 1969 ) , u s i n g a s a t u r a t e d s o l u t i o n of Sudan b l a c k B i n 70% e t h y l a l c o h o l and f r e s h f r o z e n s e c t i o n s ; the S c h u l t z method f o r c h o l e s t e r o l w h i c h a l s o r e q u i r e s f r e s h , f r o z e n s e c t i o n s ; the p e r f o r m i c - a c i d S c h i f f method f o r l i p i d s c o n t a i n i n g u n s a t u r a t e d bonds ( u s i n g f o r m a l i n f i x e d , p a r a f f i n s e c t i o n s ) ; the M i l l o n r e a c t i o n f o r t y r o s i n e ( B a k e r ' s m o d i f i c a t i o n ) ; the f e r r i c f e r r i c y a n i d e method f o r s u l p h y d r y l ( u s i n g f o r m a l i n f i x e d , p a r a f f i n s e c t i o n s and f r e s h , f r o z e n s e c t i o n s ) ; the B i e b r i c h s c a r l e t method f o r b a s i c n u c l e o p r o t e i n s a t pH l e v e l of 9.5 ( f o l l o w i n g f i x a t i o n i n C a r n o y ' s f l u i d ) ; the B i e b r i c h s c a r l e t method of S p i c e r and L i l l i e ( L i l l i e , 1965) s t a i n i n g 60 m inutes i n 0 .01% B i e b r i c h s c a r l e t i n the M c l l v a i n e -9-c i t r i c acid-sodium phosphate buffer at pH 4.95; Heidenhain's iron hematoxylin procedure; and Mallory's phosphotungstic acid hematoxylin method (Culling, 1957). The l a t t e r three methods, the Biebrich scarlet method of Spicer and L i l l i e , Heidenhain's iron hematoxylin method, and Mallory's P.T.A.H. method were also carried out on the preputial glands of adult female albino rats (200 gm.), following formalin f i x a t i o n for 12 hours, i n order to compare the findings of these methods on the rat gland with those on the mouse gland. In addition to l i p i d droplets, Schaffer (1933) and Beaver (1959, i960) have described numerous cytoplasmic granules i n the acinar c e l l s of the rat preputial gland which are quite d i s t i n c t from the f a t droplets and are best demonstrated with iron hematoxylin or with Mallory's stain. However, the demonstration of the granules, even with these methods, i s very much dependent upon proper tissue f i x a t i o n ; 10% buffered neutral formalin renders the granules insoluble so that they r e s i s t almost every subsequent treatment whereas alcoholic and certain acid f i x a t i v e s destroy the granules (Beaver, 1959). In order to ensure the exact duplication of Beaver's (i960) technique, the Biebrich scarlet method, Heidenhain's iron hematoxylin method and Mallory's P.T.A.H. method were also carried out on rat and mouse glands which had been fixed i n 10% neutral formalin buffered by sodium acetate ( L i l l i e , 1965), as well as on those glands fixed i n sodium phosphate buffered formalin. F i n a l l y , the d i f f e r e n t i a l staining method which Beaver (i960) devised to exhibit the secretion granules -10-and fat droplets simultaneously i n frozen sections of rat preputial gland was carried out on the preputial glands of adult male and female mice and also on the glands of male and female rats (200 gm.). A second method for the simultaneous d i f f e r e n t i a l staining of both secretory products in the rat preputial gland was devised using o i l red 0 and iron hematoxylin (Heidenhain's method): 1) Fix tissue i n 10% buffered neutral formalin for 12 hours. (The buffer was sodium phosphate). 2) Cut thin frozen sections about 7u i n thickness. 3) Stain for 10 minutes with o i l red 0 i n isopropanol -dilute the stock solution of o i l red 0 (lgm. o i l red 0: 500 ml. 99% isopropanol) as follows before using: 9 ml. stock: 6 ml. d i s t i l l e d water, adding the water to the o i l red 0. Let stand 5-10 minutes, then f i l t e r twice. (These three steps follow the method of Beaver, 1960.) 4) Rinse i n d i s t i l l e d water. 5) Mordant i n a 4%> aqueous solution of ammonium iron alum at 45 C for 60 minutes. 6) Rinse i n tap water. 7) Rinse i n d i s t i l l e d water. 8) Stain i n 0.5% aqueous solution of hematoxylin at 45 C for 60 mintites. 9) Rinse, i n tap water. 10) Differentiate i n 2.5% aqueous solution of ammonium iron alum. 11) Blue i n tap water. 12) Mount i n Apathy's medium. RESULTS: f a t red; granules black. -11-RESULTS Gross Anatomy of the Adult Preputial Gland The paired preputial glands of the mouse are situated subcutaneously on each side of the midline i n the genital region of the animal. The excretory duct of each gland runs along the l a t e r a l side of the penis i n the male and along the l a t e r a l side of the c l i t o r i s i n the female to open independently beside the urethral opening. A network of blood vessels may be seen upon the dorsal surface of each gland. In the male mouse, the gland i s a well developed, flask-shaped structure; i t ranges i n length from 3 to 10 mm., and i n wet weight from 8 to 81 mg., depending on the age of the adult animal (Graph l ) . In the adult female mouse, the preputial gland i s a rather small, round structure; i t averages 2 mm. i n length and ranges from 2 to 7 mg. i n wet weight, depending also on the age of the animal (Graph 2). There i s considerable v a r i a t i o n i n the weight of preputial glands even among mice of the same age which i s p a r t i a l l y related to a similar v a r i a t i o n i n body weight among such animals (Table I I ) . Contralateral glands i n the same male animal are not always comparable by weight either, there being as much as 10 mg. difference. NOTES TO GRAPHS 1 AND 2 Each point on the graph represents the body weight (gm.) or single preputial weight (mg.) of a di f f e r e n t mouse. ¥here more than one animal of the same age was used, the graph passes through the average weight recorded for that age. At 3 weeks, the minimum (min.), maximum (max.) and average (av.) single preputial gland weights indicated on graph 1 are the values compiled from a t o t a l of 17 weanling males i n experiment I I . At 7 weeks, the min., max., and av. are the values compiled from a t o t a l of 31 adult males i n experiments I and I i . At 3 weeks, the min., max., and av. single preputial weights indicated on graph 2 are the values compiled from a t o t a l of 22 weanling females i n experiment I I . At 7 weeks, the min.;, max., and av. are the values compiled from a t o t a l of 29 adult females i n experiments I and I i . -13-GRAPH 1 a b BODY WEIGHTS AND WEIGHTS OF SINGLE GLANDS OF MALE MICE FROM 1 WEEK TO 12 MONTHS LEGEND 1 2 34 56 78 WEEKS 3 4 5 6 7 8 9 10 11 MONTHS -14-GRAPH 2 ac BODY WEIGHTS AND WEIGHTS OP SINGLE PREPUTIAL GLANDS OP FEMALE MICE FROM 1 WEEK TO 12 MONTHS LEGEND 'Body Weight ( gm. ) -•Single Preputial Weight (mg.) 40 30 20 10 A-AL i i n I I I ' I ' I ' 12 34 5678 WEEKS 3 5 6 7 8 9 MONTHS 10 11 12 TABLE II Normal v a r i a t i o n s i n body weight and weights of s i n g l e p r e p u t i a l glands* i n weanling and young a d u l t mice (^compiled from weights of p r e p u t i a l glands removed from mice of experiments I, I i , and II at onset of each experiment). BODY WEIGHTS (gm.) Status No. Minimum Maximum Average Adult M 31 18.5 27.5 23.7 Adult F 29 16.2 25.1 21.0 Weanling M 17 6.2 13.2 9.2 Weanling F 22 6.5 11.6 9.1 ABSOLUTE PREPUTIAL WEIGHT (mg.) RELATIVE PREPUTIAL WEIGHT Status No. Minimum Maximum Average (mg/lOgm body Minimum Maximum wt) Average A d u l t M 31 21.0 49.3 33.0 9.7 20.3 13.8 A d u l t F 29 0.6 3.4 2.1 0.4 1.9 1.0 Weanling M 17 1.0 3.5 1.8 1.4 3.0 1.9 Weanling F 22 0.4 1.5 0.8 0.5 1.4 1.0 -16-Microscopic Anatomy of the Young Adult Preputial Gland At 5-6 weeks, the age of sexual maturity of most mice of the Swiss s t r a i n , the preputial gland exhibits a f u l l y developed holocrine, compound alveolar structural pattern. In both the male and female mouse the parenchyma of the gland consists of densely packed a c i n i of various sizes and i n various states of a c t i v i t y , a number of which can be described i n the manner of Montagna and Noback (1946) as follows: 1) small, e p i t h e l i o i d a c i n i between the larger parenchymal a c i n i ; the c e l l s of these "primordial a c i n i " have l i t t l e cytoplasm. 2) rounded a c i n i , more uniform i n size and shape; a layer of flattened peripheral c e l l s surrounds c e l l s of increasing magnitude toward the centre of each acinus; these c e l l s have vacuolated or granular cytoplasm (Fig. 3). 3) greatly enlarged a c i n i composed of hypertrophied, vacuolated c e l l s (the vacuoles represent cytoplasmic f a t t y globules) surrounded by a layer of flattened peripheral c e l l s (Fig. 4). 4) a c i n i also composed of hypertrophied, vacuolated c e l l s which are beginning to degenerate i n the centre of the acinus; these show degenerating c e l l membranes and pyknotic nuclei (Fig. 5). 5) a c i n i i n which c e l l u l a r degeneration i s well under way; a d i s t i n c t lumen appears i n the acinus; often at this -17-s tage s e v e r a l a c i n i have f u s e d t o form g i a n t cong lomera te s of d i s i n t e g r a t i n g c e l l s . The s m a l l e r a c i n i ( t ype s 1, 2 above) are most numerous i n the p e r i p h e r y of the p r e p u t i a l g l a n d . O f t e n m i t o s e s can be seen i n the b a s a l c e l l s o f these a c i n i i n d i c a t i n g the p o s s i b i l i t y of f u r t h e r i n c r e a s e i n t h e i r c e l l number. The l a r g e a c i n i , unde r go i n g h y p e r t r o p h y and subsequent d e g e n e r -a t i o n ( t ype s 3 , 4 , 5) are most numerous i n the c e n t r e of the g l a n d . The above s t a t e s of a c t i v i t y r e p r e s e n t the c y c l e of sebum f o r m a t i o n i n the p r e p u t i a l g l a n d , f rom the f a t - f r e e a c i n u s to the h y p e r t r o p h i e d , f a t - l a d e n a c i n u s i n w h i c h c e l l d e g e n e r a t i o n i s t a k i n g p l a c e . The s m a l l p r i m o r d i a l a c i n i ( t ype l ) r e p l a c e those a c i n i w h i c h have a l r e a d y formed sebum. Each a c i n u s i n c r e a s e s i n s i z e w i t h the c o n t i n u e d s t o r a ge of f a t t y s ub s tance s i n the a c i n a r c e l l s . Then, the h y p e r t r o p h i e d c e n t r a l c e l l s b e g i n t o d e g e n e r a t e . The n u c l e i become p y k n o t i c , and c e l l u l a r membranes i n d i s t i n c t . C e l l u l a r breakdown p r o g r e s s e s f rom the c e n t r e of the a c i n u s toward the p e r i p h e r y . The f l a t t e n e d p e r i p h e r a l c e l l s o f the a c i n u s , meanwh i l e , become s t r a t i f i e d t o form the e p i t h e l i a l l i n i n g (2-3 l a y e r s t h i c k ) of a new a c c e s s o r y d u c t i n the g l a n d , w h i c h , i n o r d e r t o open i n t o a duc t a l r e a d y p r e s e n t i n the g l a n d , must b reak t h r ough the s t r a t i f i e d e p i t h e l i u m of the o l d d u c t . The edges of the e p i t h e l i u m a t t h i s s i t e become f r a y e d and are c a s t o f f i n t o the sebum. E v e n t u a l l y the l i n i n g of the new a c c e s s o r y duc t becomes c on t i nuou s w i t h -18-that of the old duct, permitting the passage of the sebum from the degenerated acinus to the central duct of the gland. The complete sequence of a c t i v i t y i n the cycle of sebum formation is not limited to the uniform types of a c i n i just described. It i s seen, too, i n the large conglomerates of a c i n i i n which there is a gradual accumulation of l i p i d droplets i n the acinar c e l l s , progressing from the periphery to the centre of the a c i n i , near a duct, where c e l l u l a r degeneration, subsequently, takes place (Fig. 6). The cycle of holocrine secretion and duct formation, which has been described above, has been termed "ductular transformation" by Beaver (i960). This seems to be a useful descriptive term to denote active secretion and w i l l therefore be used throughout t h i s paper to refer to the acinar cycle. The pattern of ducts i n the preputial gland i s always changing as new l a t e r a l or accessory ducts are formed. The l a t e r a l ducts open into the central duct, which i n the 5 week old mouse i s most extensive i n the centre of the gland. The central duct i s lined by s t r a t i f i e d , squamous epithelium (4-6 layers thick) which i s not keratinized. I t , in turn, connects with the main excretory duct of the gland which i s lined by s t r a t i f i e d squamous epithelium (about 8-10 layers thick) continuous with that of the epidermis of the surrounding genital region. The squamous epithelium of the main excretory duct is for the most part keratinized (like the epidermis), and thus keratohyaline granules, stained -19-h e a v i l y by h e m a t o x y l i n , can be seen i n the s u p e r f i c i a l l a y e r s of f l a t t e n e d e p i t h e l i a l c e l l s ( P i g . 15). The main e x c r e t o r y duct i s not k e r a t i n i z e d d i s t a l l y , near i t s u n i o n w i t h the c e n t r a l d u c t . The p r e p u t i a l g l a n d i s surrounded by a conspicuous c o n n e c t i v e t i s s u e c a p s u l e w h i c h i s composed of c o l l a g e n o u s f i b r e s , f i b r o b l a s t s , macrophages and adipose t i s s u e . I t i s c o n t i n u o u s w i t h the c o n n e c t i v e t i s s u e network or stroma w i t h i n the g l a n d i t s e l f , w h ich d e l i c a t e l y surrounds each a c i n u s and u n d e r l i e s the e p i t h e l i u m of each d u c t . O c c a s i o n a l l y , t h i c k e r masses of i n t e r s t i t i a l c o n n e c t i v e t i s s u e d i v i d e the parenchyma i n t o i n d e f i n i t e l o b e s . The i n t e r s t i t i a l t i s s u e i s a l s o r i c h l y v a s c u l a r i z e d ; t h e r e are numerous c a p i l l a r i e s . I n b o t h the male and female g l a n d a s i n g l e h a i r f o l l i c l e i s i n t i m a t e l y a s s o c i a t e d w i t h the g l a n d , i n t e r -s t i t i a l l y . The h a i r f o l l i c l e can be f o l l o w e d t h r ough s e r i a l s e c t i o n s and i t s s t r u c t u r e d i s t i n g u i s h e d e i t h e r i n c r o s s -s e c t i o n or l o n g i t u d i n a l s e c t i o n , near the d i s t a l end of the main e x c r e t o r y duct surrounded by the parenchyma of the g l a n d . The s t r u c t u r e i s l i k e t h a t of any h a i r f o l l i c l e of the s k i n . An o u t e r r o o t s heath surrounds the c e l l s of the i n n e r r o o t s heath w h i c h , i n t u r n , surrounds the h a i r s h a f t . The h a i r s h a f t p r o j e c t s i n t o the p o s t e r i o r end of the c e n t r a l d u ct near i t s u n i o n w i t h the main e x c r e t o r y duct ( F i g . 7 ) . -20-B e s i d e s the obvious g r o s s d i f f e r e n c e s i n s i z e and w e i g h t (Graphs 1, 2) t h e r e are c e r t a i n sex d i f f e r e n c e s i n the s t r u c t u r a l p a t t e r n of the p r e p u t i a l g l a n d s of 5 week male and female mice. I n the male, the duct system has become e x t e n s i v e by d u c t u l a r t r a n s f o r m a t i o n ( P i g . l ) . C o n v e r s e l y , i n the female the duct system i s l e s s w e l l developed ( P i g . 2). F u r t h e r m o r e , i n the f e m a l e , the f i b r o u s i n t e r s t i t i a l t i s s u e w h i c h surrounds the a c i n i i s f r e q u e n t l y t h i c k e r than i n the male c o u n t e r p a r t ( F i g . 8). Development and A g i n g I n the newborn male and female mouse each p r e p u t i a l g l a n d c o n s i s t s of s e v e r a l masses of p r i m o r d i a l a c i n a r c e l l s i n the s u r r o u n d i n g c o n n e c t i v e t i s s u e of the prepuce ( F i g . 9). At t h i s stage t h e r e i s no d e f i n i t e c o n n e c t i v e t i s s u e c a p s u l e . There are m i t o t i c c o n f i g u r a t i o n s i n a few of the a c i n a r c e l l s . Other c e l l s i n the c e n t r e of the g l a n d are l a d e n w i t h f a t t y d r o p l e t s and have begun to degenerate by a p r o c e s s s i m i l a r t o t h a t which t a k e s p l a c e i n the a c i n i of the a d u l t g l a n d . The lumen of the d i s t a l p o r t i o n of the main e x c r e t o r y duct i s d e r i v e d from the d e g e n e r a t i o n of these h y p e r t r o p h i e d c e l l s ( F i g . 10). An i n v a g i n a t i o n of the e p i d e r m i s a c t u a l l y g i v e s r i s e to the main e x c r e t o r y duct so t h a t the s t r a t i f i e d squamous e p i t h e l i u m of the duct (which i s a l s o k e r a t i n i z e d ) m a i n t a i n s c o n t i n u i t y w i t h the s u r f a c e e p i t h e l i u m ( F i g . 10). The lumen of the extreme p r o x i m a l p o r t i o n of the duct r e s u l t s from the desquamation or -21-s l o u g h i n g o f f of e p i t h e l i a l c e l l s a t t h i s s i t e . A s i n g l e l a y e r of c u b o i d a l c e l l s w hich seems t o be co n t i n u o u s w i t h the c u b o i d a l c e l l s of the e p i t h e l i u m of the e x c r e t o r y duct surrounds the p r i m o r d i a l a c i n a r c e l l s . At b i r t h the i n t i m a t e a s s o c i a t i o n between the p r e p u t i a l g l a n d and a h a i r f o l l i c l e i s p r e s e n t ( F i g . 11). The h a i r f o l l i c l e l i e s n ext to the p r i m o r d i a l a c i n a r c e l l s and the h a i r s h a f t emerges i n t o the d i s t a l end of the main e x c r e t o r y duct ( F i g . 1 2 ) as i t does i n the a d u l t g l a n d . The o u t e r r o o t sheath c e l l s of the h a i r f o l l i c l e are con t i n u o u s w i t h the c u b o i d a l - s h a p e d , p e r i p h e r a l c e l l s of the g l a n d . T h i s i s not s u r p r i s i n g s i n c e a l l these s t r u c t u r e s are d e r i v a t i v e s of the s k i n . D u r i n g the f i r s t week of development of the p r e p u t i a l g l a n d , i n the male and female mouse a f t e r b i r t h , t h e r e i s a g r a d u a l i n c r e a s e i n s i z e , as the r e s u l t of the p r o l i f e r a t i o n , by m i t o s i s , of the p r i m o r d i a l a c i n a r c e l l s of the g l a n d . By the end of the f i r s t week, the g l a n d s are d e f i n i t e l y i d e n t i f i a b l e s t r u c t u r e s g r o s s l y . M i c r o s c o p i c a l l y , r e o r g a n i -z a t i o n of the g l a n d toward the a d u l t s t r u c t u r a l p a t t e r n b e g i n s . I n o t h e r words, some of the a c i n a r c e l l s are grouped t o g e t h e r and v a g u e l y resemble the type 1 and 2 a c i n i of the a d u l t g l a n d ; i r r e g u l a r l y shaped b a s a l c e l l s ( p r o b a b l y d e r i -v a t i v e s of the c u b o i d a l - s h a p e d p e r i p h e r a l c e l l s of the younger gland) s u r r o u n d a c i n a r c e l l s w h i c h are i n v a r i o u s degrees of enlargement. I n d i v i d u a l c e l l s w i t h i n o t h e r a c i n a r g roupings are un d e r g o i n g h y p e r t r o p h y or d e g e n e r a t i o n - 2 2 -t o fo rm sebum ( F i g . 1 3 ) . Between the se " p r i m i t i v e " a c i n i t h e r e i s a s t roma of f i b r o u s c o n n e c t i v e t i s s u e composed of many f i b r o b l a s t s and macrophages. A t 2 weeks of age the p r e p u t i a l g l a n d i s a compact s t r u c t u r e , m i c r o s c o p i c a l l y . I n b o t h the male and fema le g l a n d , the a c i n i a re now d e n s e l y packed t o g e t h e r , more u n i f o r m l y shaped, and r e c o g n i z a b l e as the s t r u c t u r a l t y pe s of the a d u l t g l a n d ( F i g . 1 4 ) . The p e r i p h e r a l c e l l s o f t he a c i n i a re now q u i t e f l a t t e n e d . Cong lomerates of a c i n i i n v a r i o u s s t age s of d u c t u l a r t r a n s f o r m a t i o n can a l s o be s een . A s t roma of d e l i c a t e c o n n e c t i v e t i s s u e , i n t e n s e l y e o s i n o p h i l i c , su r rounds the a c i n i and i s now con t i nuou s w i t h the c o n n e c t i v e t i s s u e c ap su l e around the whole g l a n d . As development c o n t i n u e s d u r i n g the i n t e r v a l f rom 3 to 5 weeks , the g l and s of b o t h sexes i n c r e a s e i n s i z e bu t the male g l a n d undergoes the more s t r i k i n g s i z e change. M i c r o s c o p i c a l l y , i n b o t h the male and fema le g l a n d , the e n l a r g e d a c i n i composed of h y p e r t r o p h i e d , v a c u o l a t e d c e l l s ( type 3) become more and more numerous. D u c t u l a r t r a n s f o r m a t i o n i s e x t e n s i v e so t h a t e v e n t u a l l y the c e n t r a l d u c t of the g l a n d l i n e d w i t h s t r a t i f i e d squamous e p i t h e l i u m opens i n t o the main e x c r e t o r y duc t (by 3 weeks) and l a t e r a l d u c t s appear i n the subs tance of the g l a n d (by 4 w e e k s ) . F i n a l l y , the g l and s a t t a i n the mature h i s t o l o g i c a l p a t t e r n ( a t 5 t o 6 weeks) p r e v i o u s l y d e s c r i b e d and the s e x u a l -23-d i f f e r e n c e s become a p p a r e n t . D u r i n g the i n t e r v a l f rom 5 t o 8 weeks , the amount o f parenchyma i n the g l a n d of the male mouse i n c r e a s e s s u b s t a n t i a l l y bu t the s t r o m a l f ramework remains d e l i c a t e . A t the same t ime the duc t system becomes more e x t e n s i v e by d u c t u l a r t r a n s f o r m a t i o n and the p a t t e r n of l a t e r a l duc t s i s c o n s t a n t l y changed by the f o r m a t i o n of new ones . By week 8, i n the c e n t r a l p o r t i o n of the g l a n d , because of the i n c r e a s e i n the duc t system and e s p e c i a l l y i n the s i z e of the lumen of the c e n t r a l d u c t , the a c i n i a re m a i n l y c o n f i n e d to the p e r i p h e r y of the g l a n d ; t h a t i s , a s m a l l a r e a between the c o n n e c t i v e t i s s u e c ap su l e and the s t r a t i f i e d e p i t h e l i u m of the d u c t s ( F i g . 1 6 ) . I n the fema le g l a n d , d u r i n g the i n t e r v a l f rom 5 to 8 weeks , the amount of parenchyma does no t change g r e a t l y (as i n the male) bu t the s t r o m a l framework becomes more abundant : f i b r o u s c o n n e c t i v e t i s s u e t r a b e c u l a e are p r o m i n e n t . D u c t u l a r t r a n s f o r m a t i o n i s g e n e r a l l y e x t e n s i v e and as the a c i n i r e g r e s s toward the p e r i p h e r y of the g l a nd t h e r e i s a compensatory i n c r e a s e i n the lumen of the c e n t r a l d u c t . Mo reove r , because of the s m a l l e r s i z e of the female g l a n d , t h e r e are few l a t e r a l d u c t s ( F i g . 17) u n l i k e the p a t t e r n i n the male g l a n d . The p e r i o d of m i c r o s c o p i c deve lopment and o r g a n i z a t i o n of the p r e p u t i a l g l a n d , f rom b i r t h u n t i l 8 weeks when a c t i v e s e c r e t i o n ( d u c t u l a r t r a n s f o r m a t i o n ) becomes r e a l l y e x t e n s i v e , i s r e f l e c t e d g r o s s l y by a p e r i o d of s t eady and pronounced g rowth of the g l a n d by w e i g h t f rom week 1 t o week 8. Graphs - 2 4 -1 and 2 g i v e the w e i g h t s ( i n mg.) f o r s i n g l e p r e p u t i a l g l and s of male and fema le mice r e s p e c t i v e l y as the ages of the a n i m a l s change. I t i s i m p o r t a n t t o note t h a t these graphs do not show a d e f i n i t i v e g rowth cu rve f o r the p r e p u t i a l g l a n d of mice bu t m e r e l y i l l u s t r a t e the g e n e r a l g rowth p a t t e r n of the g l a n d i n the male and f o r c ompa r i s on , i n the f e m a l e . The i n c r e a s e i n w e i g h t f rom week 1 t o week 8 i s c o n s i d e r a b l y g r e a t e r i n the male t h a n i n the female g l a n d . The p r e p u t i a l g l and s of o l d e r a n i m a l s have a d i f f e r e n t s t r u c t u r a l p a t t e r n as t hey undergo c e r t a i n age changes. The most s t r i k i n g age changes i n b o t h the male and fema le g l and s are of cour se the c o n t i n u o u s dec rea se i n a c i n a r t i s s u e ( F i g s . 18 and 19 ) , ( t h e r e i s no r e g e n e r a t i o n of a c i n a r t i s s u e once the a c i n u s has comp le ted i t s c y c l e ) and the compensatory i n c r e a s e i n the lumen of the c e n t r a l d u c t as the sebum c y c l e c o n t i n u e s and the l a t e r a l d u c t s are g r a d u a l l y d e s t r o y e d and c a s t o f f i n t o the sebum t o o . I n the male g l a n d , f rom 3 months t o 10 months, a compact mass of a c i n i of numerous t ype s and v a r i o u s s i z e s rema ins l o c a t e d i n the p o s t e r i o r p a r t of the g l a n d . But a n t e r i o r l y as the s u b e p i t h e l i a l stroma and i n t e r s t i t i a l t i s s u e become more abundant , the few r e m a i n i n g a c i n i a re o f t e n i n v aded by c o n n e c t i v e t i s s u e e l e m e n t s . A few l ymphocy te s o f t e n appear i n the c o n n e c t i v e t i s s u e s t roma and some f a t c e l l s accumu la te i n the i n t e r -a c i n a r t i s s u e . But the e x a c t t ime of appearance of t he se -25-l a t t e r age changes depends on the individual animal. Fat c e l l accumulation i n the interacinar tissue and lymphocytic invasion of the stroma were observed i n the preputial glands of 5, 8, and 9 month old males but not i n 6 or 7 month old animals which did not even have the abundant stroma, characteristic of aging glands. In 11 and 12 month old male mice, the preputial glands are c y s t i c ; the central and l a t e r a l ducts are f i l l e d with a substance r i c h i n lymphocytes, polymorphonuclear neutrophiles and desquamated duct e p i t h e l i a l c e l l s (Figs. 20 and 22). A c i n i , stroma, and duct epithelium degenerate as they are invaded by numerous lymphocytes. Only a few isolated a c i n i remain at the periphery and i n the posterior portion of the gland near the main excretory duct. The s t r a t i f i e d squamous epithelium of the main excretory duct does not seem to be affected by the lymphocytes. In the female gland at 3 months, the decrease i n acinar tissue, accompanied by the increase i n the size of the duct system, i s already at an advanced stage. The l i t t l e remaining parenchyma at the periphery of the gland around the ducts consists only of small a c i n i ; there are no enlarged, heavily vacuolated a c i n i and no conglomerates of a c i n i (these are s t i l l seen i n the male gland at this age). During the i n t e r v a l from 3 months to 12 months, the parenchyma continues to decrease and degenerate; the lumen of the central duct becomes larger as the l a t e r a l ducts are engulfed by i t ; f a t c e l l s accumulate i n the connective - 2 6 -t i s s u e s t roma w h i c h becomes more and more abundant . U n l i k e the male g l a n d s , the o l d e r female g l and s d i d no t become c y s t i c ( F i g . 21 ) . D e s p i t e the d e g e n e r a t i v e phenomena w h i c h accompany a g i n g , the w e i g h t s o f the p r e p u t i a l g l and s do not dec rea se w i t h age i n e i t h e r the male or the fema le as Graphs 1 and 2 i l l u s t r a t e f o r each sex r e s p e c t i v e l y . The we i gh t of the fema le g l a n d (Graph 2) rema ins f a i r l y s t e a d y d u r i n g the i n t e r v a l f rom 8 weeks t o 12 months and a l t h o u g h the we i gh t of the male g l a n d (Graph l ) f l u c t u a t e s w i d e l y d u r i n g t h i s i n t e r v a l , depend ing on the i n d i v i d u a l a n i m a l , i t never d e c l i n e s t o a v a l u e l e s s t han t h a t of the average young a d u l t ma l e . I n f a c t the g r e a t e s t w e i g h t s a re r e c o r d e d f o r 5 t o 9 month o l d g l and s i n w h i c h age changes are p r o m i n e n t . Perhaps the i n c r e a s e i n i n t e r a c i n a r t i s s u e e lements compen-s a t e s f o r the l o s s of a c i n a r t i s s u e . I n c o n c l u s i o n , a s t udy of we i gh t cannot r e v e a l the changes w h i c h o ccu r n o r m a l l y i n the p r e p u t i a l g l and s of a g i n g a n i m a l s . On ly a h i s t o l o g i c a l s t udy can show t h e s e . -27-Physiology Data on preputial gland weights were collected for each animal i n the experimental groups i n order to study the possible effect of hormonal stimulation on this physical measurement. As described previously i n the section on Materials and Methods, the two glands from each mouse of the adult and weanling groups (I, I i , and II) were weighed singly so that a comparison of weights, before and after treatment, for each animal could be made. However, such a comparison could not be made in the newborn group ( i l l ) since the paired glands from each mouse of this l a t t e r group had to be weighed together after treatment and then averaged. The average weights of single male glands and of single female glands i n each subgroup of groups I, I i , II and III were calculated, so that i t was possible to make a comparison of average weights between the subgroups within a group. In addition to the measurement of preputial gland weights, the body weight of each animal was measured before (I, I i , and II animals) and after the experiment ( a l l groups). This made i t possible to determine, for each mouse, the r e l a t i v e preputial gland weight which denotes mg. of gland per 10 gm. of body weight. Relative preputial gland weight was considered to be a more meaningful measurement than absolute preputial gland weight because of the normal range of v a r i a t i o n which i s found among mice of the same age and which seems to be p a r t i a l l y related to a similar v a r i a t i o n - 2 8 -i n body w e i g h t among such a n i m a l s (Tab le I I ) . Tab le I I I g i v e s the average r e l a t i v e w e i g h t s of s i n g l e p r e p u t i a l g l and s ( a l l g roups ) c omp i l ed f rom the d a t a , t hu s c o l l e c t e d . The d a t a were a l s o a n a l y s e d u s i n g a computer program f o r a one-way a n a l y s i s o f v a r i a n c e , w h i c h used S c h e f f e ' s t e s t f o r m u l t i p l e compar i sons w i t h unequa l sample s i z e , t o t e s t the t r e a t m e n t means. Th i s t e s t p r o v i d e d the F v a l u e s f rom wh i ch i t was p o s s i b l e t o de te rm ine the s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t subgroups . The r e s u l t s of the program are summarized i n Tab le IV. W i t h r e f e r e n c e t o Tab le IV, i n the a d u l t c a s t r a t e group I, the average r e l a t i v e w e i g h t change of the g l and s i s d i s t i n c t l y p o s i t i v e a f t e r the a d m i n i s t r a t i o n of t e s t o s t e r o n e p r o p i o n a t e , t r e a t m e n t a , and i s t h e r e f o r e s i g n i f i c a n t l y d i f f e r e n t f rom the average r e l a t i v e we i gh t changes of g l and s of the c o n t r o l subgroups d and c_, and the subgroup b_ ( a f t e r e s t r a d i o l t r e a t m e n t ) f o r b o t h male and fema le mice w i t h i n the g roup . But t h e r e i s no s i g n i f i c a n t d i f f e r e n c e between t r e a t m e n t s b, c_, and d upon the p r e p u t i a l g l a n d w e i g h t s , a l l of w h i c h p roduced an average r e l a t i v e we i gh t l o s s , w i t h the e x c e p t i o n of the fema le v e h i c l e c o n t r o l group c w h i c h showed a n e g l i g i b l e we i gh t g a i n anyway. I n the i n t a c t a d u l t group I i t h e r e were no s i g n i f i c a n t d i f f e r e n c e s between t r e a t m e n t groups f o r male mice bu t compar ing t h i s group t o males of the a d u l t c a s t r a t e group I, i t i s i n t e r e s t i n g t o note t h a t the a d m i n i s t r a t i o n of e s t r a d i o l b e n z o a t e , t r e a t m e n t b_, e f f e c t e d a s i m i l a r -29-TABLE I I I AVERAGE RELATIVE WEIGHTS OP SINGLE PREPUTIAL GLANDS Sex R e l a t i v e I n i t i a l R e l a t i v e F i n a l Treatment and P r e p u t i a l Weight P r e p u t i a l Weight Group Subgroup No. (mg/lOgm) (mg/lOgm) a)Testosterone M 5 14.6 18.0 propionate F 5 1.1 8.0 i ; b ) E s t r a d i o l M 5 12.7 7.5 c a s t r a - benzoate F 3 1.2 1.0 ted a d u l t c)Sesame O i l M 5 13.4 4.8 mice U.S.P. F 5 0.9 1.0 d) C o n t r o l M 2 14.5 6.2 F 4 1.2 1.0 a)Te stosterone M 3 12.8 13.1 propionate F 3 0.5 11.8 i i ; b ) E s t r a d i o l M 3 11.9 7.0 i n t a c t benzoate F 2 0.6 0.3 ad u l t mice •c) Sesame O i l M 1 13.3 16.2 U.S.P. F 1 1.9 0.7 d) C o n t r o l M 7 15.6 14.2 F 6 1.1 1.6 a)Te stosterone M 3 2.0 16.9 propionate F 5 1.0 9.2 i i ; b ) E s t r a d i o l M 4 1.9 1.4 i n t a c t benzoate F 5 0.9 0.9 wean-l i n g c)Sesame O i l M 5 1.8 14.2 mice U.S.P. F 6 0.8 1.1 d) C o n t r o l M 5 2.2 10.7 F 6 0.9 1.4 -30-TABLE I I I cont. Sex R e l a t i v e I n i t i a l R e l a t i v e P i n a l Group Treatment Subgroup and No. P r e p u t i a l Weight (mg/lOgm) P r e p u t i a l Weight (mg/lOgm) a)Te stosterone M 4 5.5 propionate F 6 4.0 I I I ; b ) E s t r a d i o l M 3 1.2 i n t a c t benzoate F 4 1.6 newborn mice c)Sesame O i l M 4 1.8 U.S.P. F 5 1.3 d ) C o n t r o l M 17 — — _ 2.0 F 22 0.9 The f i g u r e s f o r the c o n t r o l s i n group I I I are compiled from the average i n i t i a l r e l a t i v e p r e p u t i a l weights of a l l mice i n group I I . NOTES TO TABLE IV denotes male; F denotes female See Table I f o r f u r t h e r d e t a i l s The f i r s t f i g u r e i n each b l o c k of group I , I i , and I I r e s u l t s i s the average r e l a t i v e w e i g h t g a i n or l o s s (mg/lOgm) per p a i r of g l a n d s : t h i s f i g u r e was d e r i v e d i n the f o l l o w i n g manner u s i n g the p a i r e d glands from each a n i m a l . The r e l a t i v e w e i g h t of the g l a n d removed at. the onset of the experiment was s u b t r a c t e d from the r e l a t i v e w e i g ht of the g l a n d removed a t the end of the experiment; the sum of these d i f f e r e n c e s i n each t r e a t m e n t subgroup was d i v i d e d by the t o t a l number of a n i m a l s i n the subgroup t o g i v e the average r e l a t i v e w eight g a i n or l o s s as g i v e n i n the t a b l e . The second f i g u r e i n each b l o c k of r e s u l t s ( a l l groups) r e p r e s e n t s the s t a n d a r d e r r o r . The t h i r d f i g u r e i n each b l o c k of r e s u l t s ( a l l groups) i s the sample s i z e ; t h a t i s , the number of a n i m a l s i n the subgroup. The f i r s t f i g u r e i n each b l o c k of group I I I r e s u l t s i s the average r e l a t i v e f i n a l p r e p u t i a l g l a n d w e i g h t (mg/lOgm). The average r e l a t i v e weight g a i n or l o s s per p a i r of g l a n d s was not c a l c u l a t e d f o r t h i s group s i n c e the l e f t p r e p u t i a l g l a n d was not removed a t the b e g i n n i n g of the experiment as i n groups I , I i , and I I . T h e r e f o r e w e i g h t s b e f o r e and a f t e r each t r e a t m e n t c o u l d not be compared. I n s t e a d the p a i r e d g l a n d s from each mouse of group I I I were weighed t o g e t h e r and t h e n averaged. The f i g u r e s f o r the c o n t r o l s i n group I I I are c o m p i l e d from the average i n i t i a l r e l a t i v e p r e p u t i a l w e i g h t s of a l l mice i n group I I . i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from t r e a t m e n t a a t the 5% l e v e l , i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from t r e a t m e n t a a t the 1% l e v e l , i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from t r e a t m e n t b a t the 5% l e v e l , i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from t r e a t m e n t b a t the 1% l e v e l , i n d i c a t e s s i g n i f i c a n t d i f f e r e n c e from t r e a t m e n t c a t the 5% l e v e l . TABLE IV COMPUTER PROGRAM ANALYSIS OF THE PHYSIOLOGICAL STUDY T r e a t m e n t 3 , Group Young I : c a s t r a t e d a d u l t mice Group I i : I n t a c t Young a d u l t mice Group I l r i n t a g t Wean l i n g M i ce Group I I I : I n t a c t Newborn a m i c e M F M F M F M F a )Te s t o - 3 . 4 b 6 . 9 b 0.3 11.3 14 .9 . 8.2 5 . 5 e 4 . 0 e s t e r o n e p r o p i o -na te ± 1 . 5 C ( 5 ) d ± 0 . 8 ° ( 5 ) d ±3 .8 (3) ±2 .5 (3) ± 0 . 5 (3) ± 0 . 5 (5) ± 0 . 6 (4) 0.3 (6) b E s t r a - - 5 . 2 * - 0 . 2 * * - 5 . 0 - 0 . 3 * * - 0 . 4 * * - 0 . 0 4 * * 1 . 2 * * 1 . 6 * * d i o l b en zoa te ±0 . 5 (5) ± 0 . 2 (3) ± 3 . 1 (3) ± 0 . 1 (2) ± 0 . 2 (4) ± 0 . 1 (5) ± 0 . 3 (3) ± 0 . 3 (4) c)Sesame - 8 . 6 * * 0 . 0 3 * * 1 2 . 4 ' ' 0 . 2 * * 1 . 8 * * 1 . 3 * * O i l , U . S . P . v e h i c l e c o n t r o l ± 2 . 2 (5) ± 0 . 1 (5) ± 1 . 0 (5) ± 0 . 2 (6) ± 0 . 2 (4) ± 0 . 1 (5) d ) C o n t r o l - 8 . 3 * - 0 . 2 * * - 1 . 5 0 . 5 * * 8 . 5 * * , z 0 . 5 * * f * * 1.9 ' t ! f 0.9 ' ± 2 . 6 ± 0 . 1 ± 1 . 1 ±0 .3 ±0 .7 ± 0 . 2 ± 0 . 1 ±0 .05 (2) (4) (7) (6) (5) (6) (17) (22) -33-average r e l a t i v e weight loss i n both groups (-5.2 mg/lO gm for group IbM; -5.0 mg/lO gm for group IibM). Furthermore there i s a s t r i k i n g difference between the average r e l a t i v e weight changes of the control groups; the castrated controls show a much greater weight loss. For the females of the intact adult group I i , the administration of testosterone propionate effected a d i s t i n c t weight gain i n the glands of subgroup a (11.3 mg/lO gm) which i s s i g n i f i c a n t l y d i f f e r e n t at the 1% level from the weight changes of glands of treatment subgroups b and d. In the weanling group II the average re l a t i v e weight gain of male preputial glands (14.9 mg/lO gm) after the administration of testosterone propionate i s s i g n i f i c a n t l y d i f f e r e n t from the weight changes of male control glands (8.5 mg/lO gm) and of estr a d i o l treated glands (-0.4 mg/lO gm), but i s not s i g n i f i c a n t l y d i f f e r e n t from the average r e l a t i v e weight gain (12.4 mg/lO gm) which also occurs i n glands of the vehicle control subgroup c. In fact there i s a s i g n i f i c a n t difference at the 5% le v e l between the weight changes of the two control subgroups which is inexplicable to me although i t i s probably not a t t r i -butable to the sesame o i l injections (since no such difference occurs between controls of the other groups, male or female). What i s more important i n the male weanling group i s the si g n i f i c a n t difference which occurs between the average r e l a t i v e weight changes i n glands of both control subgroups and the estradiol treated glands. The - 3 4 -l a t t e r show an average r e l a t i v e weight l o s s . For females i n the w e a n l i n g group the a d m i n i s t r a t i o n of t e s t o s t e r o n e p r o p i o n a t e e f f e c t e d a d i s t i n c t weight g a i n i n g l a n d s of subgroup a (8.2 mg/lO gm) which i s s i g n i f i c a n t l y d i f f e r e n t a t the Yfo l e v e l from the average r e l a t i v e w e i g ht changes i n g lands of t r e a t m e n t subgroups b, c, and d. F i n a l l y , i n the newborn group I I I , f o r b o t h males and f e m a l e s , the average r e l a t i v e f i n a l p r e p u t i a l w e i g h t of glands i n subgroup a, a f t e r the a d m i n i s t r a t i o n of t e s t o s t e r o n e p r o p i o n a t e , i s s i g n i f i c a n t l y d i f f e r e n t from the average r e l a t i v e f i n a l p r e p u t i a l w e i g ht of glands i n the c o n t r o l subgroups c, d and subgroup b ( a f t e r e s t r a d i o l t r e a t m e n t ) . But t h e r e i s no s i g n i f i c a n t d i f f e r e n c e between t r e a t m e n t s b, c, and d upon male p r e p u t i a l g l a n d w e i g h t s , and t r e a t m e n t s b an _c upon female g l a n d w e i g h t s . The s i g n i f i c a n t d i f f e r e n c e , a t the 5% l e v e l between t r e a t m e n t s b and d upon the average r e l a t i v e f i n a l p r e p u t i a l w e i g h t s of female glands i n these subgroups can be r e a d i l y accounted f o r by the body weight v a r i a t i o n s i n subgroup d anima,ls. I f these f i g u r e s are examined, the body w e i g h t s seem h i g h compared to the body w e i g h t s of female mice i n the o t h e r subgroups so t h a t the r e l a t i v e p r e p u t i a l w e i g h t s of d a n i m a l s are c o r r e s p o n d i n g l y a f f e c t e d . U s u a l l y , but not a l w a y s , the weight changes i n the glands of each t r e a t m e n t subgroup are i n d i c a t i v e of t h e i r -35-present physiological state and microscopic structure. Therefore during the consideration of the microscopic appearance of; the treated preputial glands which follows, I s h a l l constantly refer to Table IV and the relationship between physical measurements and histology or physiology. Group I; castrated adult mice  Male s The l e f t preputial glands which were removed from each 7 week male mouse at the onset of the experiment exhibit the f u l l y developed, compound alveolar structural pattern (Fig. 23) which was described i n d e t a i l i n a previous section. Of course, there are s l i g h t individual variations in preputial gland size, i n the number of accessory ducts which course through the parenchyma of the gland and i n the amount of interacinar and subepithelial connective tissue. In the 10 week castrated control animals (subgroups c_, d) the preputial glands show some degree of atrophy (Fig. 24). Generally there i s a decrease in the overall size of the glands. In. the l i t t l e remaining parenchyma i t is s t i l l possible to distinguish the various types of a c i n i which make up the cycle of sebum formation but there are generally fewer c e l l s than usual i n each acinus (Fig. 28). The duct system i s also less extensive. Furthermore there i s an increase i n the stromal framework of the gland, manifest by an abundant fibrous interacinar -36-and subepithelial stroma and numerous fibrous trabeculae. A l l these structural changes give the glands a somewhat female-like microscopic pattern and indicate an overall depression of secretory a c t i v i t y which also explains the very definite post castration decrease i n r e l a t i v e weight of the gland (Tables III and.IV). The da i l y administration of testosterone propionate for three weeks to castrated male mice maintains the adult structural pattern of the preputial gland and thereby counteracts the post castration preputial glandular atrophy which was observed i n the control glands; hence the signi-r-f i c a n t difference between the control subgroups -c_ and d and a (after testosterone treatment) on the basis of r e l a t i v e weight (Table IV). But s t r u c t u r a l l y the treated glands are not stimulated by the hormone to the same degree. In three of the treated glands the stimulatory effect i s manifest microscopically by hyperplasia of the acinar c e l l s and by increased ductular transformation of tb,e parenchyma. The l a t t e r i s so extensive, that i n spite of the hyperplasia, the a c i n i are mainly confined to the periphery of the gland between the epithelium of the ducts and the connective tissue capsule (Fig. 26). The remaining two glands i n t h i s treatment group are cystic (Fig. 27), resembling the aging 11 and 12 month old male glands. The central and l a t e r a l ducts are f i l l e d with sebum, lymphocytes, polymorphonuclear neutrophiles, and desquamated duct e p i t h e l i a l c e l l s . A c i n i , -37-stroma and even duct epithelium degenerate as they are invaded by numerous lymphocytes (Fig. 29). At the periphery of the gland only a few compact masses of a c i n i remain. Here the cystic atrophy i s probably "the result of overstimulation by the repeated action of the hormone during the three week in j e c t i o n period which leads to what Beaver (i960) c a l l s "exhaustion atrophy"; hyperplasia of the acinar c e l l s cannot keep up with the c e l l u l a r destruction which of necessity accompanies holocrine secretion. F i n a l l y , i n castrated male mice after the d a i l y administration of es t r a d i o l benzoate for three weeks, the preputial glands show a similar degree of atrophy as do the control glands (of subgroups £ and d). The glands are reduced i n size both grossly, on the basis of r e l a t i v e weight (Table IV), and microscopically; there i s l i t t l e remaining parenchyma which generally consists of small a c i n i , an abundant fibrous interacinar and subepithelial stroma and a less extensive duct system (Fig. 25.). Since these changes, which give the glands the appearance of aging female glands, and indicate a depression of secretory a c t i v i t y , also occur i n the glands of male castrated controls, they may be attributable mainly to the absence of the testes rather than to the presence of the estrogenic hormone. However, there are further changes i n the estradiol treated glands of castrated males which are perhaps d i r e c t l y effected by the hormone; the thickened s t r a t i f i e d epithelium of the l a t e r a l and central ducts; the presence of many - 3 8 -desquamated e p i t h e l i a l c e l l s i n the ducts (Fig. 30). Females The l e f t preputial glands which were removed from each 7 week female mouse at the onset of the experiment exhibit, with s l i g h t individual variations, of course, the t y p i c a l structural pattern of the young adult female gland (Fig. 31) which has been previously described. After castration, the 10 week female glands i n subgroups c_ and d are not atrophic (Fig. 32), unlike the glands of male castrated controls. In the female, gross (on the basis of r e l a t i v e weight) and microscopic differences are neg l i g i b l e . A few obvious aging effects are observed but even these vary from animal to animal; a decrease i n the amount of parenchyma, smaller a c i n i , a more abundant stroma, the invasion of some a c i n i by connective tissue elements, for example. After the da i l y administration of testosterone propionate for three weeks to castrated female mice, the preputial glands show a s i g n i f i c a n t increase i n average r e l a t i v e weight (Table IV). Microscopically, the stimulatory effect of the hormone i s manifest by hyperplasia of the paren-n chymal c e l l s and by hypertrophy of the a c i n i . Therefore the a c i n i appear larger than i n the 7 week female glands or i n the 10 week control glands. Furthermore, the frequenpy of conglomerates of a c i n i (types 4 and 5) which are undergoing degeneration, has increased; the duct system has become - 3 9 -more e x t e n s i v e by d u c t u l a r t r a n s f o r m a t i o n , and o n l y a d e l i c a t e s t roma of c o n n e c t i v e t i s s u e su r rounds the a c i n i ( F i g s . 35 and 3 6 ) . Because o f these s t r u c t u r a l changes , w h i c h are the r e s u l t of t e s t o s t e r o n e s t i m u l a t i o n , the female g l and s b e g i n t o resemble the more a c t i v e l y s e c r e t i n g g l and s of i n t a c t a d u l t male m i c e . A f t e r e s t r a d i o l t r e a t m e n t the g l and s o f f ema le c a s t r a t e d mice g e n e r a l l y resemble the g l and s of the! u n t r e a t e d c o n t r o l female 's ( i n subgroups £ and d) ; the obv iou s a g i n g e f f e c t s are o b s e r v e d - t h e r e d u c t i o n i n the amount of parenchyma, the i n c r e a s e i n the s t r o m a l network ( F i g . 3 3 ) . But u n l i k e the c o n t r o l g l and s and no t u n l i k e the c o n d i t i o n i n the e s t r a d i o l t r e a t e d g l and s of c a s t r a t e d male a n i m a l s , t h e r e are changes i n t he s t r u c t u r e of the e s t r a d i o l t r e a t e d female g l and s wh i ch are perhaps d i r e c t l y e f f e c t e d by the hormone; the d u c t u l a r e p i t h e l i u m i s t h i c k e n e d and the e p i t h e l i a l c e l l s seem to be c o n t i n u o u s l y s l oughed o f f i n t o the d u c t s ( F i g . 3 4 ) . Group I i ; i n t a c t a d u l t mice The p r e p u t i a l g l and s of i n t a c t , a d u l t a n ima l s of group I i se rve as a source of c ompa r i s on , a f t e r and rogen i c and e s t r o g e n i c t r e a t m e n t s , t o the g l and s o f the s i m i l a r l y t r e a t e d c a s t r a t e d an ima l s of group I. I n c o n t r a s t t o the po s t c a s t r a t i o n g l a n d u l a r a t r o p h y i n the c o n t r o l males of group I, the p r e p u t i a l g l and s of -40-group I i male controls continue to exhibit the mature microscopic pattern (Fig. 37). A few aging effects may be observed - the lymphocytic i n f i l t r a t i o n of the connective tissue stroma, the invasion and subsequent degeneration of some a c i n i and the epithelium of some ducts by lymphocytes and other connective tissue elements. Similar results are observed i n the glands of group I i control females which, also l i k e the glands of group I control females, continue to exhibit the mature female microscopic pattern (Fig. 41). Again aging effects may be observed - a decrease i n the amount of parenchyma, an increase i n the lumen of the central duct, smaller a c i n i , a more abundant stroma, the invasion of some a c i n i by connective tissue elements (Fig. 42). After the administration of testosterone propionate to intact male animals the preputial glands show negligible gross differences on the basis of r e l a t i v e weight (Table IV) and when compared to control glands, almost negligible structural differences. The l a t t e r , however, are d i f f i c u l t to assess objectively since noticeable variations i n the amount of parenchyma and the extent of the duct system, for example, occur among the individual glands of t h i s treatment subgroup. There does seem to be an indica t i o n of increased secretory a c t i v i t y i n one of the glands, as manifest by hyperplasia of the acinar c e l l s and increased ductular transformation of the parenchyma (Fig. 38.). In contrast to t h i s , the stimulation of female glands i n intact animals -41-r e c e i v i n g t e s t o s t e r o n e i s s i g n i f i c a n t l y g r e a t e r b o t h g r o s s l y , on the b a s i s of r e l a t i v e w e i g h t (Tab le IV) and m i c r o s c o p i c a l l y . The s t i m u l a t o r y e f f e c t o f the hormone i n the female g l a n d i s m a n i f e s t m i c r o s c o p i c a l l y , i n much the same way as i n the g l and s of group l a c a s t r a t e d f ema le s - by h y p e r p l a s i a of the parenchymal c e l l s , h y p e r t r o p h y of the a c i n i , and by i n c r e a s e d d u c t u l a r t r a n s f o r m a t i o n , a l l o f w h i c h g i v e the g l a n d a m a l e - l i k e s t r u c t u r a l p a t t e r n ( F i g . 4 3 ) . There i s a re semblance between the male g l and s of group I and those of group I i a f t e r e s t r a d i o l benzoate t r e a t -ment. A l t h o u g h group I i a n i m a l s a re i n t a c t , the male g l and s are a t r o p h i c t o o , showing a s t r i k i n g dec rea se i n s i z e g r o s s l y (on the b a s i s of r e l a t i v e we i gh t - Tab le IV) and m i c r o s c o p i c a l l y ( F i g . 39) t h e r e i s l i t t l e r e m a i n i n g parenchyma w h i c h g e n e r a l l y c o n s i s t s of s m a l l - s i z e d a c i n i , an abundant f i b r o u s i n t e r a c i n a r and s u b e p i t h e l i a l s t r oma , a l e s s e x t e n s i v e duc t system ( F i g . 4 0 ) . The rea son s f o r the s t r u c t u r a l s i m i l a r i t y between the g l and s of these two subgroups are o b v i o u s l y no t the same. Whereas e s t r a d i o l , i n the c a s t r a t e a n i m a l s , does not b r i n g about the a t r o p h y of the g l a n d , i n the i n t a c t a n i m a l s , e s t r a d i o l , i n the doses g i v e n i n t h i s e x p e r i m e n t , seems to have an e f f e c t upon the g l a n d , p o s s i b l y by a l t e r i n g e x i s t i n g e n d o c r i n e r e l a t i o n s h i p s . A f t e r the a d m i n i s t r a t i o n of e s t r a d i o l benzoate to i n t a c t female an ima l s i n group I i the m i c r o s c o p i c s t r u c t u r e of the p r e p u t i a l g l a nd g e n e r a l l y re semb le s t h a t of the c o n t r o l - 4 2 -g l a n d s , w i t h the e x c e p t i o n of changes perhaps d i r e c t l y or i n d i r e c t l y e f f e c t e d by the e s t r o g e n i c hormone; the m e t a p l a s i a of the squamous e p i t h e l i u m o f some d u c t s and the desquamat ion of e p i t h e l i a l c e l l s i n t o the duc t s ( F i g . 4 4 ) . Group I I : v e a n l i n g mice  Ma les The l e f t p r e p u t i a l g l and s w h i c h were removed f rom each 3 week male mouse a t the onset of the e x p e r i m e n t , are compact s t r u c t u r e s m i c r o s c o p i c a l l y w h i c h have not y e t a t t a i n e d the a d u l t h i s t o l o g i c a l p a t t e r n a l t h o u g h the a c i n i are d e n s e l y packed t o g e t h e r and a re r e c o g n i z a b l e as the s t r u c t u r a l t ype s of the a d u l t g l a n d ( type 2, w h i c h a re the most numerous, have few c e l l s ; t ype s 3, 4 , 5 have more c e l l s i n them). There are f r e q u e n t m i t o s e s i n the b a s a l c e l l s o f the a c i n i . A c e n t r a l duc t i s u s u a l l y p r e s e n t ( F i g . 4 5 ) . A t 6 weeks the g l and s o f c o n t r o l a n ima l s show a s t r i k i n g i n c r e a s e i n average r e l a t i v e we i gh t (Tab le s I I I and I V ) . M i c r o s c o p i c a l l y , the g l and s e x h i b i t the mature s t r u c t u r a l : p a t t e r n ; t h e r e i s much parenchyma composed of e n l a r g e d a c i n i o f t ype s 2, 3, 4 , 5 w h i c h now have many more c e l l s i n them and the duc t system i s e x t e n s i v e ( F i g . 4 6 ) . The e f f e c t of the d a i l y a d m i n i s t r a t i o n of t e s t o s t e r o n e p r o p i o n a t e f o r 19 days t o w e a n l i n g mice (subgroup a) i s d i f f i c u l t t o a s s e s s . G r o s s l y , t h e r e i s an i n c r e a s e i n - 4 3 -r e l a t i v e weight and s i z e but g e n e r a l l y the i n c r e a s e i s no g r e a t e r t h a n t h a t observed i n c o n t r o l g l a n d s (which i s the r e s u l t of aging) f o r , as noted p r e v i o u s l y , the average r e l a t i v e weight g a i n of t e s t o s t e r o n e t r e a t e d glands i s s i g n i f i c a n t l y d i f f e r e n t from the average r e l a t i v e w e i g ht g a i n of c o n t r o l subgroup d glands but i s not s i g n i f i c a n t l y d i f f e r e n t from the average r e l a t i v e w e i g ht g a i n of glands i n the v e h i c l e c o n t r o l subgroup £ (Table I V ) . M i c r o s c o p i c a l l y , i n some of the g l a n d s t h e r e seems to be an i n d i c a t i o n of i n c r e a s e d s e c r e t o r y a c t i v i t y as m a n i f e s t by h y p e r p l a s i a of the a c i n a r c e l l s ( F i g . 4 7 ) . The e f f e c t of e s t r a d i o l b e n z o a t e , a f t e r 19 days of t r e a t m e n t , i s , on the o t h e r hand, most n o t i c e a b l e , b o t h g r o s s l y (Tables I I I and IV) and m i c r o s c o p i c a l l y because of the s m a l l amount of parenchyma i n the glands of subgroup b a n i m a l s compared to t h a t i n c o n t r o l gLands. Furthermore the parenchyma c o n s i s t s of s m a l l a c i n i (of a l l t y p e s ) which have few c e l l s i n them. Some a c i n i are invaded by the abundant stroma w h i c h now surrounds the a c i n i and l i e s beneath the e p i t h e l i u m of the d u c t s . The duct system i s not e x t e n s i v e . As the r e s u l t of p r o l o n g e d e s t r o g e n i c t r e a t m e n t the g l a n d i s a t r o p h i c ; the normal growth p a t t e r n i s i n h i b i t e d by the hormone treatment ( F i g . 4 8 ) . Female s S t r u c t u r a l l y the l e f t p r e p u t i a l g l a n d which was removed from each 3 week female mouse a t the b e g i n n i n g of - 4 4 -the e x p e r i m e n t , r e semb le s t h a t o f the male w e a n l i n g ( F i g . 4 9 ) . But a t 6 weeks a f t e r s e x u a l d i f f e r e n c e s have appea red , the g l and s of c o n t r o l f ema le s show o n l y a s l i g h t i n c r e a s e i n s i z e . M i c r o s c o p i c a l l y , the g l and s e x h i b i t the mature s t r u c t u r a l p a t t e r n of the f e m a l e ; g e n e r a l l y the a c i n i have many more c e l l s i n them and cong lomera te s of a c i n i , wh i ch are unde rgo i ng d i s s o l u t i o n ( type 5) a re more numerous; the c e n t r a l duc t and a c c e s s o r y duc t s a re w e l l deve l oped and may occupy the c e n t r e of the g l a n d so t h a t the parenchyma i s c o n f i n e d t o the p e r i p h e r y of the g l a n d ; f i n a l l y , the i n t e r a c i n a r f i b r o u s c o n n e c t i v e t i s s u e and the f i b r o u s t r a b e c u l a e are more abundant ( F i g . 50 ) . A f t e r the a d m i n i s t r a t i o n of t e s t o s t e r o n e p r o p i o n a t e f o r 19 days t o w e a n l i n g fema le mice ( t r e a t m e n t a ) , the p r e p u t i a l g l ands undergo a s t r i k i n g i n c r e a s e i n r e l a t i v e w e i g h t and s i z e compared t o the c o n t r o l g l and s (Tab le I V ) . The s t i m u l a t o r y e f f e c t i s a g a i n m a n i f e s t m i c r o s c o p i c a l l y by h y p e r p l a s i a o f the parenchymal c e l l s , h y p e r t r o p h y of the a c i n i , and by i n c r e a s e d d u c t u l a r t r a n s f o r m a t i o n , a l l of w h i c h g i v e the g l a nd a m a l e - l i k e s t r u c t u r a l p a t t e r n ( F i g . 51 ) . E s t r a d i o l b e n z o a t e , a f t e r 19 days of t r e a t m e n t , has a s i m i l a r e f f e c t m i c r p s c o p i c a l l y upon the female g l ands o f group I I a n ima l s as i t does upon the male g l ands i n t h i s g roup. The parenchyma c o n s i s t s of s m a l l a c i n i (of a l l t y p e s ) w h i c h have few c e l l s i n them. A v e r y abundant s t roma - 4 5 -su r rounds the a c i n i ; some a c i n i a re i n v aded by the c o n n e c t i v e t i s s u e e lements of the s t r oma . The d u c t s a re not v e i l d e v e l o p e d ; the d u c t u l a r e p i t h e l i u m i s o f t e n t h i c k e n e d and e p i t h e l i a l c e l l s seem t o be c o n t i n u o u s l y s l oughed o f f i n t o the duc t s ( F i g . 52 ) , Group I I I : newborn mice The p r e p u t i a l g l and s wh i ch were removed i n i t i a l l y f rom the male and female mice of group I I se rve as the c o n t r o l g l and s of group I I I a n ima l s a t 3 weeks. A few newborn an ima l s r e c e i v e d a d a i l y i n j e c t i o n of sesame o i l f o r 3 weeks to serve as v e h i c l e c o n t r o l s . M i c r o s c o p i c a l l y , t h e n , the p r e p u t i a l g l and s of a l l the group I I I c o n t r o l a n i m a l s , b o t h male and f e m a l e , have the immature s t r u c t u r a l p a t t e r n of the w e a n l i n g mouse; a compact mass o f a c i n i ( g e n e r a l l y s m a l l i n s i z e bu t r e c o g n i z a b l e as the t ype s of the a d u l t g l and ) w h i c h su r rounds a s m a l l c e n t r a l d u c t . The d a i l y a d m i n i s t r a t i o n of t e s t o s t e r o n e p r o p i o n a t e to newborn mice f o r 3 weeks has a s i g n i f i c a n t e f f e c t upon the average r e l a t i v e w e i g h t of the p r e p u t i a l g l ands of b o t h male and female mice (Tab le I V ) . M i c r o s c o p i c a l l y , i n b o t h male and female g l a n d s , the s t i m u l a t o r y e f f e c t i s m a n i f e s t by h y p e r p l a s i a of the parenchymal c e l l s and i n c r e a s e d d u c t u l a r t r a n s f o r m a t i o n of the parenchyma as i n d i c a t e d by the p re sence of many cong lomera te s of type 4 _46-and 5 a c i n i and by the appearance of l a t e r a l ducts (Figs. 53 and 54). In the majority of the treated glands the most numerous a c i n i are s t i l l the small type 2 a c i n i which are t y p i c a l l y present i n the control glands. However, two of the treated male glands show a greater response to the hormone, for there has not only been hyperplasia of the acinar c e l l s but hypertrophy of many of the a c i n i as well (Fig. 58) so that the glands begin to exhibit the adult structural pattern. After the d a i l y administration of estradiol benzoate to newborn mice for 3 weeks, the male and female preputial glands continue to resemble, generally, the normal glands of weanling mice, with the exception of a few structural d e t a i l s , which are not widespread; the more abundant stroma, the thickened epithelium l i n i n g the central duct, the invasion of some a c i n i by connective tissue elements (Figs 55 and 56). Summary of the effects of the physiological treatments The mouse preputial gland responds d i r e c t l y to testosterone propionate. The stimulatory effect i s manifest microscopically most often by hyperplasia of the parenchymal c e l l s and occasionally by hypertrophy of the aci n i (usually i n the female glands). Therefore mitoses, -47-p a r t i c u l a r l y i n the w e a n l i n g and newborn a n i m a l s (groups I l a and I l i a r e s p e c t i v e l y ) are f r e q u e n t i n the b a s a l a c i n a r c e l l s . S t i m u l a t i o n of the female glands i s g r e a t e r t h a n t h a t of the male glands i n a l l subgroups r e c e i v i n g t e s t o s t e r o n e w i t h the e x c e p t i o n of the newborn group. The mouse p r e p u t i a l g l a n d i s not s t i m u l a t e d by e s t r a d i o l benzoate. The a c t u a l response of the g l a n d to the hormone depends on the age of the a n i m a l . A f t e r p r o l o n g e d treatment of newborn ani m a l s (group I l l b ) the m i c r o s c o p i c appearance of the p r e p u t i a l g l a n d s i s l i t t l e a f f e c t e d compared to t h a t of normal a n i m a l s of the same age. The glands of b o t h subgroups e x h i b i t the o v e r a l l immature p a t t e r n . But the glands of w e a n l i n g animals r e c e i v i n g e s t r a d i o l (group l i b ) do not develop n o r m a l l y . The e f f e c t i s more n o t i c e a b l e i n male glands because d u r i n g the i n t e r v a l from 3 weeks to 6 weeks, when n o r m a l l y male glands show evidence of a c t i v e s e c r e t i o n , the growth of male gl a n d s r e c e i v i n g e s t r a d i o l i s i n h i b i t e d ; the glands become a t r o p h i c . S i m i l a r l y , the glands of i n t a c t a d u l t males r e c e i v i n g e s t r a d i o l (group l i b ) show an o v e r a l l d e p r e s s i o n of s e c r e t o r y a c t i v i t y m a n i f e s t by d e c r e a s e d d u c t u l a r t r a n s f o r m a t i o n and a c i n a r a t r o p h y a l l of which g i v e the glands the appearance of a g i n g female g l a n d s . A d d i t i o n a l e f f e c t s of e s t r a d i o l - the t h i c k e n i n g of the d u c t u l a r e p i t h e l i u m and the c o n t i n u o u s s l o u g h i n g o f f of - 4 8 -e p i t h e l i a l c e l l s i n t o the d u c t s , f o r example, "which are noted i n some glands a f t e r e s t r a d i o l t r e a t m e n t s - w i l l be d i s -cussed i n the f o l l o w i n g s e c t i o n of the t h e s i s . H i s t o c h e m i s t r y The l i p i d component of the mouse p r e p u t i a l g l a n d i s most c l e a r l y demonstrated i n f r e s h f r o z e n s e c t i o n s of t i s s u e by the f a t - s o l u a b l e dye, Sudan b l a c k B, used as a s a t u r a t e d s o l u t i o n i n 70% e t h y l a l c o h o l . C o l o u r e d b l u e -b l a c k by the dye j numerous l i p i d d r o p l e t s appear s c a t t e r e d t h roughout the cytoplasm o f the a c i n a r c e l l s . The d r o p l e t s seem to accumulate g r a d u a l l y i n the a c i n i , p r o -g r e s s i n g from t i n y d r o p l e t s i n the s m a l l a c i n i , to l a r g e spheres i n the h y p e r t r o p h i e d and d e g e n e r a t i n g a c i n i , and f i n a l l y , t o homogenous masses of l i p i d i n the ducts of the g l a n d ( F i g . 6 0 ) . ( Q u a l i t a t i v e d i f f e r e n c e s i n the l i p i d component of a d u l t and w e a n l i n g g l a n d s , of e i t h e r sex, were not n o t i c e a b l e . ) The r e s u l t s of McManus' Sudan b l a c k B method f o r compound l i p i d s ( p h o s p h a t i d e s and c e r e b r o s i d e s ) were not c o n s i s t e n t . I n c o u n t e r s t a i n e d s e c t i o n s , t h e r e were sometimes i n the s m a l l a c i n i a few f a t d r o p l e t s , p a r t i a l l y s t a i n e d b l a c k and i n the hy p e r -t r o p h i e d a c i n i , c l o s e l y packed g l o b u l e s w h i c h f i l l e d each a c i n a r c e l l almost c o m p l e t e l y . These, t o o , were o n l y -49-heavily outlined by the stain; they did not appear as the dense droplets described above by the other Sudan technique (Fig. 6 l ) . Sudan black B has a strong a f f i n i t y for most classes of l i p i d s so that positive results with McManus' method do not necessarily s i g n i f y the presence of phosphatides and cerebrosides. Only i f neutral fats are excluded from the l i p i d component does the s t a i n become useful i n the demonstration of these compound l i p i d s (Pearse, 1968). However, neutral fats cannot be excluded from the l i p i d component of the mouse preputial gland as shown by the positive results of o i l red 0 staining by the d i f f e r e n t i a l staining technique of Beaver (i960). O i l red 0 i s a strong colourant of fats but a very poor one for phospholipids (Chayen, Bitensky, Butcher, Poulter, 1969). In the mouse preputial gland the concentration of fats i s very great as manifest by o i l red 0 staining. Moreover, the deposition of o i l red 0 droplets follows closely that revealed by Sudan black B staining i n frozen sections; the o i l red 0 droplets are conspicuous throughout the gland with the largest droplets coalescing i n the degenerating acinar c e l l s of the hypertrophied a c i n i (Figs. 62 and 67). The l i p i d component of the gland did not sta i n following the performic-acid Schiff method for compound l i p i d s containing unsaturated bonds (that i s , for - 5 0 -phospholipids and cerebrosides); there was only diffuse staining of the cytoplasm and rather more intense staining of the nuclei (the l a t t e r because of t h e i r DNA content). The Schultz method for cholesterol and i t s esters was also negative. The several histochemical tests carried out on the mouse preputial gland for proteins did not reveal d e f i n i t e structures of a proteinaceous nature i n the acinar c e l l s , which would contribute d i s t i n c t l y to the formation of the sebum, i n addition to the l i p i d component, as the c e l l s degenerated. In b r i e f , after the Millon reaction for proteins containing tyrosine and after the Biebrich scarlet method for basic nucleoproteins (at pH 9.5) there was only diffuse staining of the parenchyma and the stroma of the gland. The f e r r i c ferricyanide method which should reveal the presence of sulphur containing amino acids (cystine and cysteine) by the formation of the pigment, Prussian blue, gave ri s e to weak and non-specific green staining throughout the parenchyma of the gland. However i n sections of the gland which also included the epidermis of the prepuce, and the extreme proximal portion of the main excretory duct of the gland, the squamous epithelium of these structures stained p o s i t i v e l y , because of their keratin content. (Keratin contains S-S and S-H groups). The intensity of -51-reaction i n the epithelium of the main excretory duct decreased rapidly from the surface inwards. The Biebrich scarlet method of Spicer and L i l l i e (at pH 4.95), Heidenhain's iron hematoxylin method and Mallory's phosphotungstic acid hematoxylin method gave rise only to non-specific diffuse staining i n the acinar c e l l s of the mouse preputial gland (Fig. 66). In contrast, these methods demonstrated i n the parenchyma of the rat preputial gland, the characteristic cytoplasmic, proteina-ceous granules, previously described by Beaver (1959, 1960). There were no differences between the phosphate formalin fixed and the acetate formalin fixed rat glands. With Biebrich scarlet at pH 4.95, the granules, which were mainly perinuclear i n position and f a i r l y uniform i n size (Fig. 63), stained bright red; with iron hematoxylin the granules stained black, rea d i l y and intensely (Fig. 65): with phosphotungstic acid hematoxylin, they stained blue-black, i n contrast to the red staining of the stroma and the pale blue staining of the nuclei (Fig. 64). The granules are always most numerous and large i n size i n the acinar c e l l s approaching degeneration. In the ducts they seem to coalesce to form large, intensely staining masses of secretion. Following the d i f f e r e n t i a l staining technique of - 5 2 -Beaver (i960) upon the mouse preputial gland, only o i l red 0 droplets were observed throughout the parenchyma and as expected on the basis of the procedures described above, no perinuclear granules were observed. However, i n the rat gland both l i p i d droplets and protein granules were demonstrated simultaneously with this method (Fig. 68). Within the acinar c e l l s of the rat gland and most conspicuously, within the c e l l s of the hypertrophied a c i n i , the intensely blue-staining granules primarily surround and hence obscure the nucleus; the o i l red 0 droplets have a more random d i s t r i b u t i o n . The ducts of the gland are f i l l e d with large o i l red 0 droplets and clumps of blue stained material of considerable size which must be formed by the coalescence of many protein granules. Unfortunately, with Beaver's method, many detail s of the protein granules within the a c i n i were obscured by strong cytoplasmic and stromal d e t a i l , brought about by the use of hematoxylin as the counterstain. For this reason a second method for the simultaneous d i f f e r e n t i a l staining of both secretory products i n the rat preputial gland was devised using o i l red 0 and iron hematoxylin, as described i n the section on Materials and Methods. This second method c l e a r l y demonstrates the d i s t r i b u t i o n of protein granules (black) and f a t droplets (red) within the gland (Fig. 69). Moreover, i t documents the de t a i l s obscured by Beaver's method -- 5 3 -the development of the protein granules within the a c i n i from tiny granules i n the small a c i n i to larger spheres i n the hypertrophied a c i n i to masses of considerable size i n the ducts. Unfortunately, what was gained with one secretory product was l o s t with the other; the int e n s i t y of, o i l red 0 staining was not as sati s f a c t o r y with this second method. DISCUSSION H i s t o l o g y D e v e l o p m e n t a l l y and h i s t o l o g i c a l l y the p r e p u t i a l g l ands of the mouse are e x c e s s i v e l y deve l oped sebaceous g l and s (Vos s , 1932 ) . F u n c t i o n a l l y , t h e y may have the c h a r a c t e r of c e r t a i n a c c e s s o r y sex g l a n d s , and e i t h e r se rve as the source of a n a t u r a l l u b r i c a n t d u r i n g c o p u l a t i o n or se rve as the source or s i t e of a s e x u a l a t t r a c t a n t (B ronson , 1966; Gaunt , 1967 ) . The l a t t e r v i e w i s the f a v o u r e d one and w i l l be g i v e n f u r t h e r c o n s i d e r a t i o n l a t e r i n t h i s d i s c u s s i o n . Bo th Voss (1932) and S c h a f f e r (1933 ) , who g i v e b r i e f d e s c r i p t i o n s o f the p r e p u t i a l g l ands i n the norma l a d u l t male mouse ( n e g l e c t i n g those of the f e m a l e ) , r e c o g n i s e t h e i r r e l a t i o n s h i p t o the o r d i n a r y sebaceous g l ands of the s k i n . These a u t h o r s emphasize the i n t i m a t e a s s o c i a t i o n of the p r e p u t i a l g l a n d w i t h a h a i r f o l l i c l e w h i c h p e r s i s t s t h r o u g h o u t the l i f e t i m e of the a n i m a l . A t y p i c a l sebaceous g l a n d , w h i c h i s a p e a r - s h a p e d , s imp le or b ranched h o l o c r i n e , a l v e o l a r g l a n d , u s u a l l y d i f f e r e n t i a t e s f rom the c e l l s of the e x t e r n a l r o o t s hea th of a g row ing h a i r f o l l i c l e - The s h o r t duc t of the sebaceous g l a n d , t h u s , opens i n t o the h a i r c a n a l so t h a t the s t r a t i f i e d e p i t h e l i a l c e l l s o f the duc t are c on t i nuou s w i t h the o u t e r r o o t s hea th c e l l s of the h a i r f o l l i c l e . The e p i t h e l i u m of the duct i s , i n t u r n , continuous with the p e r i p h e r a l l a y e r of c u b o i d a l c e l l s of the sebaceous gland a l v e o l u s . The sebaceous c e l l s of the a l v e o l u s c o n t i n u o u s l y a r i s e and d i f f e r e n t i a t e from the c e l l s of the p e r i p h e r a l l a y e r to become larg e s p h e r i c a l c e l l s engorged w i t h cytoplasmic l i p i d d r o p l e t s . The f a t t y s e c r e t i o n of the sebaceous gland i s then produced by the d i s i n t e g r a t i o n of these hypertrophied c e l l s i n the r e g i o n of the duct (Odland, 1966). The p r e p u t i a l gland more c l o s e l y resembles the simple sebaceous gland i n the neonatal mouse than i n i t s l a t e r stages of development. The p r i m o r d i a l c e l l s are, at t h a t time, surrounded by a s i n g l e l a y e r of cu b o i d a l c e l l s which seems to be continuous w i t h the c u b o i d a l c e l l s of the e p i t h e l i u m of the e x c r e t o r y duct, as i n the o r d i n a r y sebaceous gland. The h a i r f o l l i c l e , which i s i n close p r o x i m i t y to the a c i n a r c e l l s , i s a l r e a d y a f u l l y developed s t r u c t u r e . However, i n c o n t r a s t to the o r d i n a r y sebaceous g l a n d - h a i r f o l l i c l e complex, the r e l a t i o n s h i p between the h a i r f o l l i c l e and p r e p u t i a l gland has under-gone a s h i f t i n emphasis. The long e x c r e t o r y duct, through which the h a i r s h a f t e x i t s , i s more c l e a r l y a s s o c i a t e d w i t h the p r i m o r d i a l gland (here the l a r g e r of the two s t r u c t u r e s ) than i t i s a s s o c i a t e d w i t h the h a i r f o l l i c l e as a h a i r c a n a l . Furthermore, the formation of the -56-excretory duct of the preputial gland involves d i s t a l l y , the degeneration of hypertrophied, fat-laden acinar c e l l s and proximally, the desquamation of e p i t h e l i a l c e l l s . But, i n mouse f o l l i c l e s of the skin, sebaceous c e l l s have not been observed to take a part i n the formation of the hair canal which actually results only from the ker a t i n i z a t i o n of c e l l s i n the epidermis (Hardy, 1949). In f e t a l l i f e , the duct of the preputial gland i n i t s early development as a primordium, may be more d i r e c t l y related to the formation of the hair f o l l i c l e . Unfortunately, a study of the development of the preputial gland i n f e t a l mice was not undertaken for this thesis. Such a study would prove interesting i n determining the i n t r i c a c i e s of the association between the preputial gland and the hair f o l l i c l e prior to b i r t h and the time of i n i t i a t i o n of these structures i n the embryo. Regarding the l a t t e r , i t i s known i n the mouse that the period of development for first-formed f o l l i c l e s , from the stage when the primordium i s f i r s t seen to the stage when the hair emerges at the skin surface, i s nine days (Lyne, 1966). Since the hair f o l l i c l e of the preputial gland i s f u l l y formed i n the newborn mouse, the anlagen of the hair f o l l i c l e and i t s associated preputial gland probably appears some nine days e a r l i e r , at the end of the second week of f e t a l l i f e . ¥ith the establishment of the compound alveolar pattern previously described, the greatly enlarged preputial gland of the adult mouse bears l i t t l e resemblance to the -57-simple sebaceous gland. The formation of p r e p u t i a l gland sebum s t i l l r e s u l t s from a d i s i n t e g r a t i o n of h y p e r t r ophied c e l l s , a h o l o c r i n e type of s e c r e t i o n . Conglomerates of a c i n i , i n which there i s a gradual accumulation of l i p i d d r o p l e t s i n the c e l l s p r o g r e s s i n g from the p e r i p h e r y to the centre of the a c i n i nearest the ducts (as i n the sebaceous gland a l v e o l u s ) , do e x i s t throughout the a d u l t p r e p u t i a l gland. But, the uniform a c i n i , i n v a r i o u s s t a t e s of a c t i v i t y i n the c y c l e of sebum formation, the types of my previous d e s c r i p t i o n , are numerous too. The ever-changing p a t t e r n of ducts, which r e s u l t s from the breakdown of hypertrophi e d a c i n i and the subsequent s t r a t i f i c a t i o n of t h e i r p e r i p h e r a l c e l l s , i s a unique f e a t u r e of the p r e p u t i a l gland. U n l i k e the sebaceous gland, i n which new c e l l s are c o n s t a n t l y produced at the p e r i p h e r y of the a l v e o l u s , there i s no r e g e n e r a t i o n of a c i n a r c e l l s i n the p r e p u t i a l gland, once the acinus has formed sebum. New a c i n i can a r i s e only, by m i t o s i s , from the small e p i t h e l i o i d a c i n i which are found between the l a r g e r parenchymal a c i n i . As the animal ages, and the p r i m o r d i a l a c i n i become depleted, the most s t r i k i n g age change of the p r e p u t i a l gland i s the o v e r a l l decrease of a c i n a r t i s s u e accompanied by the increase i n the s i z e of the duct system. The p a i r e d p r e p u t i a l glands of the r a t are s i m i l a r i n most respects to those of the mouse. Th e i r subcutaneous p o s i t i o n , i n both male and female r a t s , on each side of the -58-m i d l i n e i n the g e n i t a l r e g i o n of the a n i m a l , i s the same as t h a t i n the mouse. However, u n l i k e the obvious s i z e d i f f e r e n c e w hich e x i s t s between the glands of the a d u l t male and female mouse, the glands of r a t s are almost as e q u a l l y w e l l d eveloped i n the female as i n the male (Buschke, 1933; Beaver, I 9 6 0 ) . The glands of the a d u l t male mouse and the a d u l t male r a t d i f f e r to a c e r t a i n e x t e n t i n e x t e r n a l appearance; i n the mouse, the g l a n d s t e n d t o be more f l a s k - s h a p e d ; i n the r a t , t h e y more resemble a f l a t t e n e d c l u b . M i c r o s c o p i c a l l y , the r a t p r e p u t i a l g l a n d , l i k e t h a t of the mouse, e x h i b i t s the h o l o c r i n e , compound a l v e o l a r s t r u c t u r a l p a t t e r n w i t h a s i m i l a r c y c l e of sebum f o r m a t i o n and duct f o r m a t i o n , and s i m i l a r developmental and a g i n g h i s t o l o g y (Montagna and Noback, 1946; Beaver, I 9 6 0 ) . However, a s t r i k i n g d i f f e r e n c e i n m i c r o s c o p i c anatomy between the p r e p u t i a l g l a n d s of the mouse and the r a t i s the absence of any a s s o c i a t i o n of the r a t p r e p u t i a l g l a n d w i t h a c e n t r a l h a i r f o l l i c l e i n a d u l t l i f e or even a f t e r b i r t h as t h e r e i s i n the mouse ( S c h a f f e r , 1933; Beaver, I 9 6 0 ) . Beaver ( i 9 6 0 ) has found h a i r f o l l i c l e s i n t e r s t i t i a l l y i n r a t g l a n d s , but o n l y r a r e l y and, because t h e s e , i f f o l l o w e d by s e r i a l s e c t i o n , do not e x t e n d i n t o a d uct (as i n the mouse) but extend beyond the c a p s u l e of the g l a n d , he concludes t h a t t h e i r i n c l u s i o n w i t h i n the g l a n d i s " p r o b a b l y f o r t u i t o u s and r e l a t e d to the development of the g l a n d i n an a r e a r i c h i n h a i r f o l l i c l e s . " Physiology Since the i n i t i a l experiments of Korenchevsky and Dennison (1934a, 1934b, 1935, 1936a, 1936b) on the histology of various sex organs of the rat, including the preputial glands, after treatments with male or female sex hormones, several authors (Salmon, 1938; Noble and C o l l i p , 1941; Barnes and Swyer, 1951; Rennels, Hess, and Finerty, 1953; Huggins, Parsons, and Jensen, 1955; Beaver, 1960; Freeman, H i l f , Iovino, and Michel, 1964) have also centered their attention on the responses of the rat preputial gland to androgens, estrogens and other hormones. Their findings repeatedly show that the preputial gland i s stimulated by androgens but not by estrogens. Cursory endocrinological investigations involving the mouse preputial gland have been carried out by Voss (1932) and Burdick and Gamon (1941), whose findings establish the positive response of the mouse preputial gland to androgens. However, a detailed description of the effects of androgens and estrogens on the overall microscopic structure of the gland i n male and female mice of di f f e r e n t ages and of di f f e r e n t status (which has been presented by Beaver (i960) for the rat) i s not found i n the l i t e r a t u r e , to my knowledge. The simple sebaceous glands of the white rat respond p o s i t i v e l y to androgens and negatively to estrogens. Ebling (1948) has found that testosterone propionate ( l mg. d a i l y -60-f o r 36 days) i n c r e a s e s the a c t i v i t y and the s i z e of the sebaceous g l a n d s , (the r e s u l t o f h y p e r p l a s i a and h y p e r t r o p h y ) whereas e s t r a d i o l benzoate i n e i t h e r massive (lQO ug. d a i l y f o r 36 days) or moderate doses ( l ug. d a i l y f o r 36 days) causes a t r o p h y of the g l a n d s . H a s k i n , L a s h e r , and Rothman (1953) have a l s o shown t h a t t e s t o s t e r o n e p r o p i o n a t e t r e a t m e n t ( l mg. d a i l y f o r 30 days) i n d u c e s enlargement of the sebaceous g l a n d s (a 400% average i n c r e a s e i n s i z e ) . T h e r e f o r e , i t i s not s u r p r i s i n g t h a t the p r e p u t i a l glands r e a c t a c c o r d i n g l y when t r e a t e d w i t h these hormones. I n i n t a c t a d u l t and w e a n l i n g mice r e c e i v i n g t e s t o s t e r o n e p r o p i o n a t e ( d a i l y f o r 3 weeks), the s t i m u l a t i o n of the female glands i s s i g n i f i c a n t l y g r e a t e r t h a n t h a t of the male g l a n d , not o n l y on the b a s i s of r e l a t i v e weight but a l s o m i c r o s c o p i c a l l y , as m a n i f e s t by h y p e r p l a s i a of the a c i n a r c e l l s , the h y p e r t r o p h y of the a c i n i and by i n c r e a s e d d u c t u l a r t r a n s f o r m a t i o n , a l l of which g i v e the female g l a n d a m a l e - l i k e s t r u c t u r a l p a t t e r n . I n newborn mice, however, male and female glands respond almost e q u a l l y t o t e s t o s t e r o n e s t i m u l a t i o n . The re a s o n f o r the d i f f e r e n c e i n r e s p o n s i v e n e s s among the a d u l t and the w e a n l i n g glands i s not c l e a r . Among a d u l t g l a n d s , a t l e a s t , i t may be due t o the f a c t t h a t i n the i n t a c t a d u l t male, s t i m u l a t i o n of the g l a n d by the anima l ' s own androgenic hormones i s . a l r e a d y maximal. A p p a r e n t l y , i n the r a t , the female glands of a l l groups -61-(newborn, immature weanling, and adult animals) respond to testosterone to a greater extent than the male glands (Beaver, I960) so that Beaver (i960) suggests that the effect i s more l i k e l y due to an inherent sex difference than to the fact that androgenic stimulation of the male gland by the animal's own hormones i s near maximal. Such an inherent sex difference may also be the important factor for the difference i n responsiveness among the adult and weanling glands of the mouse. The fact that the microscopic pattern of the preputial gland of the adult male mouse i s maintained by the presence of androgens is further i l l u s t r a t e d by the atrophic changes which occur i n the gland after castration of the adult animal and thus, i n the absence of t e s t i c u l a r androgens. The atrophy is manifest by a reversion to a non-stimulated, somewhat female-like pattern, which, however, as shown, can be counteracted by the administration of testosterone to the castrated animal. Voss (1932) too, found that testes transplants or androgen injections to 4 week castrates could reverse preputial glandular atrophy, bringing about a reappearance of the secretory a c t i v i t y of the gland. In the female mouse after castrata.cn, microscopic changes i n the structure of the preputial gland are n e g l i g i b l e ; the gland continues to exhibit the t y p i c a l non-stimulated structural pattern of the adult female. In the rat, l i k e the mouse, there is a post castration atrophy i n the male -62-g l a n d but not i n the female g l a n d (Beaver, I960). The mouse p r e p u t i a l g l a n d i s not s t i m u l a t e d by e s t r o g e n s . I n f a c t , the a c t u a l response of the g l a n d u l a r parenchyma t o p r o l o n g e d t r e a t m e n t s of e s t r a d i o l benzoate seems to depend on the age and s t a t u s of the a n i m a l ; the glands of newborn mice are l i t t l e a f f e c t e d ; the g l a n d s of w e a n l i n g mice, b o t h male and f e m a l e , show i n h i b i t e d growth; the glands of i n t a c t a d u l t males (but not of females) show a c i n a r a t r o p h y and an o v e r a l l d e p r e s s i o n of s e c r e t o r y a c t i v i t y as m a n i f e s t by d e c r e a s e d d u c t u l a r t r a n s f o r m a t i o n . The a t r o p h i c changes of the l a t t e r , however, a l s o o c c u r r e d i n the glands of c a s t r a t e d males and were a t t r i b u t e d to the absence of the t e s t e s . Perhaps i t may be c o n c l u d e d , t h e r e f o r e , t h a t the a t r o p h y of the p r e p u t i a l g l a n d w h i c h f o l l o w s the i n j e c t i o n s of e s t r a d i o l i n t o the i n t a c t a n i mal i s not due to the s u p p r e s s i o n of the g l a n d by the e s t r o g e n per se but i s due to the e s t r o g e n i c s u p p r e s s i o n of the gonado-t r o p i c f u n c t i o n of the a n t e r i o r p i t u i t a r y , w hich r e s u l t s i n a l o s s of endogenous androgen. The f a c t i s now w e l l e s t a b l i s h e d t h a t the a n t e r i o r lobe of the p i t u i t a r y , which produces the g o n a d o t r o p h i c hormone complex, FSH and LH e x e r t s a c o n t r o l l i n g i n f l u e n c e upon the gonads, the gonadal s e c r e t i o n s and hence, the a s s o c i a t e d a c c e s s o r y organs. The a c t i v i t y of the a n t e r i o r p i t u i t a r y , on the o t h e r hand, i s to some e x t e n t c o n t r o l l e d by the gonadal hormones because when p r e s e n t i n e f f e c t i v e amounts, gonadal -63-hormones, of either sex, depress the gonadotrophic secretions of the anterior p i t u i t a r y (apparently i n d i r e c t l y , by their action on certain areas of the hypothalamus that are concerned with hypophyseal a c t i v i t y ) . Moore and Price (1932), among the results of several experiments, give evidence that the i n j e c t i o n of oestrin into normal adult 1 male rats leads to t e s t i c u l a r damage, a loss of t e s t i s hormone secretion and the involution of the reproductive accessory organs (the prostate and the seminal v e s i c l e s ) . However, the i n j e c t i o n of oestrin accompanied by the administration of fresh hypophyseal implants does not have a harmful effect upon the male reproductive system. Their interpretation of these findings i s based upon the complex interrelations of the gonads and the anterior p i t u i t a r y as mentioned above and may be equally v a l i d here i n the matter of the response of the preputial gland to estradiol in intact male mice. The estradiol may so suppress the secretion of hypophyseal LH that an i n s u f f i c i e n t amount i s available for the endocrine function of the t e s t i s . In the absence of t e s t i c u l a r androgens, the preputial glands be-come atrophic, i n a manner similar to that which occurs after testes removal. However, this interpretation is complicated by the fact that steroid-gonadotrophin relationships do not always f a l l into the simple pattern outlined above; recent evidence suggests that the i n t e r -play of steroids (particularly.estrogen) and gonadotrophins, i n the female mammal, depends on the dose of estrogen -64-injected; small doses of estrogen stimulate FSH output, large doses of estrogen i n h i b i t FSH output, and moderate doses of estrogen stimulate LH release (Tepperman, 1968). In my study, the injected dose of estrogen was low (only 0.00165 mg. d a i l y ) , but i t i s impossible to say whether such a dosage, given during prolonged treatment, i s phy s i o l o g i c a l l y low, moderate, or high, for the mouse. Therefore, I am unable to suggest, with any v a l i d i t y , the steroid-gonadotrophin relationships which may exist i n my experimental mice, after the injections of e s t r a d i o l . In the intact male rat, there i s evidence that small doses of estrogen produce atrophy of the androgen-secreting, i n t e r s t i t i a l c e l l s of the t e s t i s , by d i r e c t l y i n h i b i t i n g them, whereas larger doses i n the intact animal stimulate these same c e l l s by increasing the output of hypophyseal LH (Tepperman, 1968). Assuming that the dosage of estradiol used i n my study i s , indeed, physiolo-g i c a l l y low, then a reduction i n t e s t i c u l a r androgens, brought about by the dir e c t estrogenic i n h i b i t i o n of the Leydig c e l l s , may account for the atrophy of the preputial glands of the intact adult and weanling male mice. An additional hypothesis, which cannot be e n t i r e l y eliminated as an explanation of the response of the mouse preputial gland to estrogen i s the p o s s i b i l i t y of a direct suppressive action of the estrogen on the parenchyma of the preputial gland, i t s e l f . Evidence from the rat suggests that - 6 5 -such an e x p l a n a t i o n may be u n l i k e l y ; the p r e p u t i a l glands of the female r a t are almost as w e l l developed as those of the male and a f t e r p r o l o n g e d t r e a t m e n t s of exogenous e s t r a d i o l , the female glands are not a f f e c t e d (Beaver, I 9 6 0 ) . I f e s t r o g e n , i n d e e d , a c t s d i r e c t l y and s u p p r e s s i v e l y upon the p r e p u t i a l g l a n d , one would perhaps expect the female g l a n d s of i n j e c t e d a n i m a l s to be a p p r o p r i a t e l y a f f e c t e d . However, i t i s dangerous to make e x t r a p o l a t i o n s from one sex t o the o t h e r s i n c e t h e r e may be an i n h e r e n t sex d i f f e r e n c e i n v o l v e d i n the responses of the p r e p u t i a l g l a n d s of male and female animals to the e s t r o g e n . F u r t h e r m o r e , because of s p e c i e s d i f f e r e n c e s i t i s o f t e n i n a d v i s a b l e t o e x t r a p o l a t e from one s p e c i e s t o a n o t h e r , i n t h i s i n s t a n c e , from the r a t t o the mouse. I n a d d i t i o n t o the s u p p r e s s i v e e f f e c t s of e s t r a d i o l upon the mouse p r e p u t i a l g l a n d ( e s p e c i a l l y , the male g l a n d ) , which I am unable to a t t r i b u t e to one p a r t i c u l a r mechanism of a c t i o n , t h e r e are o t h e r h i s t o l o g i c a l changes which, I t h i n k , must r e s u l t from a d i r e c t a c t i o n of e s t r o g e n on the p r e p u t i a l g l a n d . These changes - namely, the m e t a p l a s i a of the squamous e p i t h e l i u m of the main e x c r e t o r y duct and the c e n t r a l duct w i t h i n d i c a t i o n s of t h e i r k e r a t i n i z a t i o n , and the desquamation of e p i t h e l i a l c e l l s i n t o these ducts - are n o t e d , o n l y a f t e r e s t r a d i o l t r e a t m e n t , i n some glands of b o t h sexes i n the a d u l t and -66-weanling groups. Several authors give evidence of similar d i r e c t estrogenic effects i n the accessory reproductive glands of the rat and the mouse after prolonged treatment. Burrows (1935, 1937) describes the pathological conditions induced by large doses of estrogenic compounds i n the coagulating gland, the prostate, and the bulbo-urethral gland of the male mouse; these generally take the form of e p i t h e l i a l metaplasia which, i n the prostate and bulbo-urethral gland, occurs i n the ducts, f i r s t l y , and then gradually spreads through the body of the gland. In the coagulating glands, which show the most marked changes, e p i t h e l i a l metaplasia i s accompanied by keratinization; the sacs of the gland become f i l l e d with keratinized material, desquamated c e l l s and polymorpho-nuclear leucocytes which r e c a l l the c y c l i c a l phenomena of estrus i n the mouse vagina. Arai (1968) found squamous e p i t h e l i a l metaplasia i n the seminal vesicles and coagulating glands of male rats which was apparently caused by neonatal treatments of high doses of estrogen and was s t i l l present 11 months after the l a s t i n j e c t i o n . The coagulating glands also showed c o r n i f i c a t i o n l i k e those of the mouse. No direct estrogenic effects have been described i n the rat gland by Beaver (i960), a fact which i s perhaps related to the observation that estrogen effects on male accessory glands are usually more marked i n the mouse than in the rat (Price and Williams-Ashman, 1961). Beaver (i960) -67-used low doses of estrogen. It i s possible, then, that high doses of estrogen (as Ara i , 1968, used) and d i f f e r e n t periods of i n j e c t i o n would affect the rat preputial gland d i r e c t l y . Possible endocrine relationships i n developing and aging preputial glands At 5 weeks, the compound alveolar structural pattern of the mouse preputial gland i s well developed, the cycle of holocrine secretion and duct formation, active, and sexual differences between male and female glands, apparent. During the interval from 5-8 weeks, sexual divergence becomes very obvious as the male gland undergoes a tremendous change i n size and weight which i s manifest, microscopically, by a substantial increase i n the amount of parenchyma i n the gland. Holocrine secretion i s , by then, very active i n the male gland; hence there develops the elaborate and extensive pattern of ducts which is not seen i n the female gland. Coinciding with this development of the preputial gland i s the attainment of sexual maturity in the animal, i t s e l f . The precise time of sexual maturity, which depends on the proper balance and interaction of anterior p i t u i t a r y and gonadal hormones, i s highly variable i n Mus musculus (there are the usual individual variations and s t r a i n v a r i a t i o n s ) . Further-more, i t must be interpreted within the framework of the measurement used to describe sexual maturation. For example, indices of sexual maturity for the female mouse can be the - 6 8 -f i r s t appearance of the vaginal o r i f i c e , or the f i r s t appearance of the c o r n i f i e d vaginal smear (both of these are estrogen dependent), or the i n i t i a l willingness to mate, or f i n a l l y , the a b i l i t y to conceive and carry a l i t t e r to term fahich does not always occur with the schedule of the previous events); for the male, indices of sexual maturity can be the f i r s t appearance of spermatozoa i n the epididymis, or the i n i t i a l willingness to mate. Because my chief concern i s the endocrinological condition of the gonads, a d e f i n i t i o n of sexual maturity as the capacity of the gonads to function, which implies gonadal hormone a c t i v i t y but excludes any reference to behavioural cycles, i s adequate. I have not been able to find in the l i t e r a t u r e a s p e c i f i c reference to the age of Swiss albino mice at normal sexual maturity, but i n our breeding lab, a young female mouse of the Swiss s t r a i n kept with males of the same l i t t e r w i l l usually have i t s f i r s t l i t t e r at 8 weeks of age. Presumably then, sexual maturity i s attained in both sexes by the age of 5 weeks. This figure agrees well with the findings of several authors on the age of maturity i n undesignated strains of the albino mouse. According to Cockrum (1962), Mus musculus reaches maturity in 35 days (that i s , 5 weeks). Engle and Rosasco (1927) found the average age of the f i r s t appearance of the vaginal o r i f i c e i n albino mice to be 35 days with a range from day 28 to day 49 whereas Mirskaia and Crew (1930) found the mean age of f i r s t estrus to be 39 days, with male -69-mice reaching maturity at about the same time. Since the secretion of androgen controls the a c t i v i t y of the preputial gland, i t i s not surprising that the maturation of the male gland coincides with the age of sexual maturity at about 5 weeks and i s followed by the establishment of the stimulated pattern during the i n t e r v a l from 5-8 weeks. At this time androgen production i n the male mouse must be very active. In addition to the postpuberal growth of the male preputial gland there i s a prepuberal period of steady growth by weight (see graph l ) which i s manifest microscopically by increases i n the amount of glandular parenchyma. In the r a t , i t i s known that the secretory a c t i v i t y of the testes gradually increases during prepuberal development, i n response to the r i s i n g t i t e r s of c i r c u l a t i n g gonadotrophins, u n t i l the l e v e l of androgen maintained i n the adult i s reached (Moore and Price, 1938). Similar secretory a c t i v i t y by the anterior p i t u i t a r y and the developing testes i n the mouse would account for t h i s prepuberal stage of preputial gland growth. There i s evidence to support t h i s assumption. For example, i t i s known that the d i f f e r e n t i a t i o n of the mouse hypophysis from a gonadotrophic viewpoint occurs at day 6 i n males and at day 12 i n females (Bronson, Dagg, and S n e l l , 1966) and that thereafter the capacity of the gonads to respond to gonadotrophic hormone stimulation -70-develops gradually, increasing rapidly at about 3 weeks of age (Price and Ortiz, 1944). Therefore, i t i s reasonable to assume that the production of t e s t i c u l a r androgens increases gradually during the prepuberal development of the mouse, i n response to the increasing gonadotrophic a c t i v i t y . The preputial gland develops accordingly; i t s s t r i k i n g r e a c t i v i t y begins after 3 weeks of age (see graph l ) , the time, at which, i t i s interesting to note, that gonadal a c t i v i t y increases s i g n i f i c a n t l y . But what of androgen production i n the female mouse? Ye have seen that the female preputial gland, l i k e that of the male, i s stimulated by androgens but not by estrogens. The female preputial gland i s not unique i n this respect. The prostate homologue i n the female mouse responds only to androgenic stimulation too. In the female mammal the production of androgens seems to occur normally i n the ovaries and the adrenal glands (Parkes, 1950; Price and Williams-Ashman, 1961), under the influence of hypophyseal hormones. Such androgenic production i n the young female mouse, increasing gradually in response to increasing gonadotrophic a c t i v i t y , u n t i l the l e v e l of androgen maintained in the adult i s reached, would account for the prepuberal period of growth a,n& development of the female preputial gland -71-(see graph 2), up to the time of sexual maturity when the mature microscopic pattern of the gland i s reached and thereafter maintained. Androgen production i n the female mouse must, of course, be very much less than i n the male mouse. Not only i s the prepuberal growth of the female gland less s t r i k i n g than that of the male but i t s postpuberal growth i s almost ne g l i g i b l e , i n contrast to that of the male gland. It i s possible that the mouse preputial gland responds d i r e c t l y to c i r c u l a t i n g p i t u i t a r y hormones as well as to androgens. It has been demonstrated, for example, that the rat preputial gland undergoes marked atrophy following hypophysectomy (Noble and C o l l i p , 1941; Beaver, I960); an atrophy which i s not completely reversible by the administration of testosterone alone. The presence of the p i t u i t a r y gland seems to be necessary for testosterone to exert i f s f u l l action on the preputial gland. As additional evidence, Noble and C o l l i p (l94l) have shown i n female rats that p i t u i t a r y extracts can stimulate the preputial glands to a certain extent i n the absence of ovaries and adrenal glands. The stimulatory effects of the p i t u i t a r y extracts were augmented i n intact animals. Perhaps then, i t i s t h i s a c t i v i t y of the p i t u i t a r y which explains why ovariectomy does not affect the preputial gland of the adult female mouse; i t i s s u f f i c i e n t to maintain the "non-stimulated" pattern of the -72-female gland i n the absence of ovarian androgens. The adrenal androgens may also make a contribution to the maintenance of the preputial gland following ovariectomy. It i s d i f f i c u l t to assess th e i r r e l a t i v e importance because I carried out no adrenalectomy experiments. However, there are indications i n the l i t e r a t u r e that adrenal androgens can usually cause only s l i g h t effects (Price and Williams-Ashman, 1961) and i t has been shown i n female rats that the removal of both ovaries and adrenals does not aff e c t the weight of the preputial glands (Noble and C o l l i p , 1941). If the preputial gland follows the t y p i c a l pattern of development of certain mammalian reproductive accessory glands, such as the prostate (Price, 1936; Burns, 1961), i t s early development i n the male mouse before b i r t h may be dependent on the secretion of male hormone by the f e t a l t e s t i s , whereas, i n the female mouse, i t s early development may proceed independently of hormonal conditioning (up to a certain stage), But, since the preputial glands of newborn mice, of both sexes, are so similar i n size and i n microscopic structure, i t i s possible that the f e t a l development of male glands may be independent of hormonal stimulation, as well as that of female glands, u n t i l the d i f f e r e n t i a t i o n of the hypophysis from the gonadotrophic point of view, at day 6 i n males and at day 12 i n females. (It i s interesting to note that this i n i t i a t i o n of gonadotrophic a c t i v i t y i s followed closely by the growth -73-of the male gland at week 1 and of the female gland at week 2.) In order to determine the precise role of hormones i n the early development of the male preputial gland, a study of the effects of early castration on the glands of f e t a l and neonatal mice would have to be undertaken. The preputial glands of mice age quickly. During the i n t e r v a l from 3-12 months both male and female glands undergo a tremendous reduction and degeneration of acinar tissue, accompanied by an increase i n the size of the duct system. By 12 months, the glands are made up almost e n t i r e l y of a network of empty ducts. In the prostate glands of aging rats and mice (the exact age i s not stated), senile changes, similar to those brought about by castration, have been described by Moore (1936) and are interpreted on the basis of a decrease or absence of t e s t i c u l a r androgen. However, hormonal deficiencies of the gonads are probably not a notable factor i n the aging processes of the preputial gland. These age changes are not similar to the atrophic changes caused by castration. Moreover, during the int e r v a l from 3-12 months, the reproductive a c t i v i t i e s of the mouse normally continue; t h i s implies gonadal a c t i v i t y . According to Snell (1941), the useful breeding period of the inbred female mouse terminates at about 1 year of age; thereafter, there i s a decrease i n f e r t i l i t y and fecundity which is s t r a i n dependent and hence, highly variable. Actually, a gradual decrease in -74-f e r t i l i t y i n aging female mice need not necessarily r e f l e c t a lowered estrogen production by the ovaries. Endocrine a c t i v i t y can apparently be retained longer than reproductive capacity i n some mice, as shown by Thung, Boot and Miihlbock (1956) for the inbred strains C57BL, O20, and the hybrid (O20 x DBAf). Unfortunately, to my knowledge, there has been no consideration of t h i s relationship i n the Swiss s t r a i n of mice. S t i l l , for a l l these reasons given above, i t seems unlikely that endocrine deficiencies are responsible for the aging of mouse preputial glands during the interval from 3 to 12 months. More important factors i n aging must be changes i n the response of the preputial gland to androgens, once the gland has f u l l y developed (at 3 months i n some animals). Androgens must continue to stimulate the secretory a c t i v i t y of the gland but must cease to appreciably affect the regeneration of parenchyma i n the adult gland. With the gradual depletion of primordial a c i n i and the cessation of mitotic a c t i v i t y i n the acinar c e l l s , the effective " l i f e " of the preputial gland i s fixed by the amount of parenchyma which makes up the gland at this stage of development. Thereafter, the parenchyma of the gland inevitably diminishes; the result of the c e l l u l a r destruction which, of necessity, accompanies holocrine secretion. In this way, the preputial gland i s unique among the accessory reproductive glands of the 75-mouse; for, i n most accessory reproductive glands secretory a c t i v i t y is a continuous process dependent upon an androgen stimulated epithelium. Histochemistry The histochemical studies of my thesis confirm the h i s t o l o g i c a l observations on the cycle of sebum formation i n the mouse preputial gland; that, i n f a c t , the secretion of the gland is l i p i d which i s elaborated i n the c e l l s of the a c i n i and discharged into the ducts when the c e l l s degenerate. The l a t t e r could not be observed i n the h i s t o l o g i c a l preparations because of the use of the fat solvents i n the p a r a f f i n processing which dissolved the l i p i d ; therefore, the ducts always appeared empty and the acinar c e l l s , vacuolated. Much of the l i p i d secretion of the mouse preputial gland seems to be made up of neutral fats ( t r i g l y c e r i d e s ) as shown by o i l red 0 staining. Other classes of l i p i d which are possibly present in the secretion, in addition to the neutral f a t s , could not be i d e n t i f i e d by the methods of the present investigation. However, Gaunt (1967), using a s i l i c i c acid column, separated free f a t t y acids as well as neutral l i p i d s from mouse preputial gland secretion and Patterson (i960) found that the preputial glands of young male mice, when incubated i n 14 acetate, incorporated the C a c t i v e l y info sterols so that -76-these, too, may normally be present i n the l i p i d secretion. The l i p i d s of the rat preputial gland have been examined by Gallo (1966) whose findings demonstrate the following d i f f e r e n t classes: squalene, t r i g l y c e r i d e s , s t e r o l esters, monoglyderides, diglyderides, free f a t t y acids, and free sterols. Of these, squalene seems to be the major component. In contrast, squalene has not been lo c a l i z e d i n the mouse preputial gland, whose f a t t y secretion i s , therefore, more closely related to that of the skin sebaceous glands of mice and rats, i n which squalene i s replaced by A - cholestenol (Brooks, Lalic.h, Baumann, 1956). The rat preputial gland, however, in i t s preferential accumulation of squalene, resembles the functional sebaceous glands of the human dermis i n which squalene is also produced i n large amounts (Kandutsch, 1964). No s p e c i f i c proteins were l o c a l i z e d i n the acinar c e l l s of the mouse preputial gland. Thus, there exists a s t r i k i n g difference i n the histochemistry between the preputial glands of the mouse and the r a t . In the l a t t e r , in addition to the l i p i d s , the acinar c e l l s contain numerous protein granules which are sepreted along with the l i p i d . The granules have no counterpart i n the c e l l s of the simple sebaceous gland nor i n the c e l l s of the mouse preputial gland i n which a l l attempts to demonstrate them, following the methods of Beaver (1959, 1960), proved f r u i t l e s s . -77-Two functions have been proposed for the preputial gland: l ) that the gland serves as the source of a natural lubricant during copulation or 2) that the gland serves to attract members of the opposite sex. The findings of recent research indicate that the l a t t e r function i s probably the.more l i k e l y one. Bronson (1966) has demonstrated the a t t r a c t i o n of C57BL./6J female mice to the preputial gland secretion of the male. Gaunt (1967), after v e r i f i c a t i o n of t h i s a t t r a c t i o n in Swiss mice, has observed that i t i s the free f a t t y acid component of the male preputial gland secretion (and not the neutral l i p i d component) which attracts female mice and i n addition, accelerates the estrus of grouped females (the Whitten e f f e c t ) . Although the free f a t t y acid component of the preputial gland i s the site of an attractant or pheromone, the o r i g i n a l source of the pheromone and i t s i d e n t i t y remain to be determined. -78-SUMMARY AND CONCLUSIONS The p r e p u t i a l glands of the male and female mouse are p a i r e d , subcutaneous organs s i t u a t e d i n the g e n i t a l r e g i o n of the a n i m a l , one on e i t h e r s i d e of the v e n t r a l m i d l i n e . Each g l a n d opens onto the s k i n , b e s i d e the u r e t h r a l opening, v i a an e x c r e t o r y d u c t . The p r e p u t i a l g l a n d s are b a s i c a l l y e x c e s s i v e l y developed sebaceous glands and so d e v e l o p m e n t a l l y , l i k e the s i m p l e sebaceous g l a n d s , t h e y are d e r i v a t i v e s of the s k i n . F u r t h e r m o r e , each g l a n d i s i n t i m a t e l y a s s o c i a t e d w i t h a h a i r f o l l i c l e w h i c h p e r s i s t s throughout the l i f e t i m e of the a n i m a l . H i s t o l o g i c a l l y , the g l a n d s are h o l o c r i n e compound, a l v e o l a r g l a n d s . D u r i n g p o s t n a t a l development both male and female glands i n c r e a s e i n s i z e , as the r e s u l t of the p r o l i f e r a t i o n of a c i n a r c e l l s , but the male g l a n d undergoes the more s t r i k i n g enlargement. Once each a c i n u s has formed sebum, t h e r e i s no r e g e n e r a t i o n of a c i n i . T h e r e f o r e , w i t h a g i n g , as the c y c l e of h o l o c r i n e s e c r e t i o n and duct f o r m a t i o n c o n t i n u e s , the parenchyma of the a d u l t p r e p u t i a l g l a n d g r a d u a l l y d e c r e a s e s . L i p i d s , p r i m a r i l y i n the form of d r o p l e t s of n e u t r a l f a t s , w hich accumulate i n the c y t o p l a s m of the a c i n a r c e l l s , a r e , t o my knowledge, the o n l y s e c r e t o r y p r o d u c t of the mouse p r e p u t i a l g l a n d . The l i p i d i s d i s c h a r g e d i n t o the -79-ducts of the g l a n d as the a c i n a r c e l l s degenerate. The p r e p u t i a l g l a n d of the mouse i s s t i m u l a t e d by t e s t o s t e r o n e p r o p i o n a t e but not by e s t r a d i o l benzoate. The s t i m u l a t o r y e f f e c t of t e s t o s t e r o n e i s m a n i f e s t , most o f t e n , by h y p e r p l a s i a of the a c i n a r c e l l s , h y p e r t r o p h y of the a c i n i , and by i n c r e a s e d s e c r e t i o n . The degree of response depends upon the age and sex of the a n i m a l . The female gland tends to respond to exqgenous t e s t o s t e r o n e t o a g r e a t e r e x t e n t than the male g l a n d , w i t h the p o s s i b l e e x c e p t i o n of the glands of newborn a n i m a l s . The s t r u c t u r a l changes which occur i n the female g l a n d a f t e r t e s t o s t e r o n e s t i m u l a t i o n g i v e the gland a m a l e - l i k e s t r u c t u r a l p a t t e r n . S i n c e the a n d r o g e n - s t i m u l a t e d g l a n d d i f f e r s s t r u c t u r a l l y from the n o n ^ s t i m u l a t e d g l a n d , and s i n c e n o r m a l l y androgen p r o d u c t i o n must be v e r y much l e s s i n the female mouse than t h a t i n the male, i t i s not s u r p r i s i n g t o f i n d m o r p h o l o g i c a l d i f f e r e n c e s i n the glands of normal a d u l t male and female mice. Furthermore, because the m i c r o s c o p i c p a t t e r n of the male g l a n d i s m a i n t a i n e d by the presence of androgens, t h e r e i s a p o s t - c a s t r a t i o n a t r o p h y i n the male gland which i s m a n i f e s t by a r e v e r s i o n to a n o n - s t i m u l a t e d somewhat fe m a l e -l i k e p a t t e r n . I n the female mouse, a f t e r c a s t r a t i o n , m i c r o s c o p i c changes i n s t r u c t u r e of tb.e p r e p u t i a l g land are n e g l i g i b l e . P r o l o n g e d t r e a t m e n t s of e s t r a d i o l benzoate a f f e c t the p r e p u t i a l g l a n d i n d i r e c t l y and/or d i r e c t l y . Some b r i n g about - 8 0 -a c i n a r a t r o p h y and a d e p r e s s i o n of s e c r e t o r y a c t i v i t y as m a n i f e s t by d e c r e a s e d d u c t u l a r t r a n s f o r m a t i o n . However, the a c t u a l degree of response of the g l a n d u l a r parenchyma depends upon the age and s t a t u s of the a n i m a l ; the glands of i n t a c t a d u l t males (but not of females) become a t r o p h i c ; the glands of w e a n l i n g mice, both male and female, show i n h i b i t e d growth; the glands of newborn mice seem to be l i t t l e a f f e c t e d . A d i r e c t a c t i o n of e s t r o g e n on the p r e -p u t i a l g l a n d i s m a n i f e s t , i n some g l a n d s , by m e t a p l a s i a of the squamous e p i t h e l i u m of the main e x c r e t o r y d u c t , and by the desquamation of e p i t h e l i a l c e l l s i n t o these d u c t s . With r e f e r e n c e t o the hormone tr e a t m e n t s of t h i s s tudy ( w i t h e i t h e r t e s t o s t e r o n e p r o p i o n a t e or e s t r a d i o l benzoate) i t must be remembered t h a t the r e s u l t s were o b t a i n e d w i t h c e r t a i n doses, so t h a t i t i s p o s s i b l e t h a t d i f f e r e n t doses or d i f f e r e n t i n t e r v a l s of t r e a t m e n t would g i v e d i f f e r e n t r e s u l t s . . H i s t o l o g i c a l l y and p h y s i o l o g i c a l l y ( i n terms of response t o hormone t r e a t m e n t s ) the p a i r e d p r e p u t i a l glands of the mouse are s i m i l a r i n many r e s p e c t s to those of the r a t . However, t h e r e are s t r i k i n g d i f f e r e n c e s i n the m i c r o -s c o p i c anatomy and i n the h i s t o c h e m i s t r y of these homologous organs which are i n t e r e s t i n g to note: f i r s t l y , the absence of any a s s o c i a t i o n of the r a t p r e p u t i a l g l a n d w i t h a c e n t r a l h a i r f o l l i c l e a f t e r b i r t h as t h e r e i s i n the mouse; s e c o n d l y , the presence of p e r i n u c l e a r , p r o t e i n a c e o u s g r a n u l e s i n the -81-a c i n a r c e l l s of the r a t p r e p u t i a l g l a n d , wh i ch have no c o u n t e r p a r t i n the a c i n a r c e l l s of the mouse p r e p u t i a l g l a n d . D e v e l o p m e n t a l l y , h i s t o l o g i c a l l y , and h i s t o c h e m i c a l l y , the p r e p u t i a l g l and of the mouse remains a n e x c e s s i v e l y deve l oped sebaceous g l a n d . But the p r e p u t i a l g l a nd of the r a t cannot be c a t e g o r i z e d so s i m p l y , a t l e a s t h i s t o -c h e m i c a l l y . Because of i f s appa ren t a b i l i t y t o s e c r e t e two d i f f e r e n t s u b s t a n c e s , the r a t p r e p u t i a l g l and has been d e s i g n a t e d a " d i c r i n e " g l a nd ( S c h a f f e r , 1933; Beave r , 1960) , i n o r d e r t o d i s t i n g u i s h i t f rom the " m o n o c r i n e " v a r i e t y of h o l o c r i n e g l and to w h i c h be long s the mouse p r e p u t i a l g l a n d . - 8 2 -PLATE 1 - The Young A d u l t P r e p u t i a l G l and of Mus muscu lus F i g . 1. P r e p u t i a l g l a nd of t he male mouse, a t 5 weeks, showing a c t i v e s e c r e t i o n and d u c t f o r m a t i o n . 37 X. a) c e n t r a l d u c t b) l a t e r a l d u c t c) g l a n d u l a r parenchyma d) c o n n e c t i v e t i s s u e c a p s u l e F i g . 2. P r e p u t i a l g l a nd of the f e m a l e , a t 5 weeks. Compare w i t h f i g u r e 1. Note the d i f f e r e n c e i n s izje and the r e l a t i v e underdeve lopment of the d u c t s y s tem. 37 X. F i g . 3. G l a n d u l a r parenchyma - d e t a i l o f t ype 2 a c i n i , showing a l a y e r of f l a t t e n e d p e r i -p h e r a l c e l l s ( b l a c k a r row) s u r r o u n d i n g c e l l s of i n c r e a s i n g magni tude toward t he c e n t r e of each a c i n u s . The v a c u o l e s p r e s e n t i n some c e l l s , r e p r e s e n t f a t t y g l o b u l e s . 476 X. F i g . 4. A t ype 3 a c i n u s ( b l a c k a r row) composed of h y p e r t r o p h i e d , v a c u o l a t e d c e l l s , s u r r ounded by a l a y e r of f l a t t e n e d p e r i p h e r a l c e l l s . 476 X. ' . - 8 3 -PLATE 2 - The Young A d u l t P r e p u t i a l G land of Mus muscu lus P i g . 5. A t ype 4 a c i n u s whose c e n t r a l c e l l s a r e b e g i n n i n g t o degene ra te i n the c e n t r e o f the a c i n u s . The n u c l e i a re p y k n o t i c . The p e r i p h e r a l c e l l s a re becoming s t r a t i f i e d ( b l a c k a r r o w ) . 476 X. F i g . 6. D e t a i l o f t he p r e p u t i a l g l a n d of a 5 week male mouse, showing cong l omera te s of a c i n i (a ) i n wh i ch t h e r e i s a p r o g r e s s i o n o f h y p e r t r o p h i e d and d e g e n e r a t i n g c e l l s f r om the p e r i p h e r y t o the c e n t r e of the a c i n i , near a d u c t ( b ) . 146 X. F i g . 7. The h a i r f o l l i c l e (a) w h i c h i s u s u a l l y a s s o c i a t e d w i t h each p r e p u t i a l g l a n d . I t s s h a f t p r o j e c t s i n t o the p o s t e r i o r end of the c e n t r a l d u c t (b) near i t s u n i o n w i t h the main e x c r e t o r y d u c t ( c ) as shown h e r e . 91 X. F i g . 8. D e t a i l of the p r e p u t i a l g l a nd of a 5 week f ema le mouse. Compare w i t h f i g u r e 6. Note the t h i c k e n e d f i b r o u s i n t e r s t i t i a l t i s s u e ( s t roma) wh i ch su r rounds the a c i n i i n t h i s f ema le g l a n d . 2 2 8 X. - 8 4 -PLATE 3 - The Development of the P r e p u t i a l G land F i g . 9. The p r e p u t i a l g l a nd of the newborn mouse (a) i n the s u r r o u n d i n g c o n n e c t i v e t i s s u e of the p r epuce . 91 X. F i g . 10. D e t a i l of the newborn p r e p u t i a l g l a n d , showing the d e r i v a t i o n of i t s main e x c r e t o r y d u c t . 228 X. F i g . 11. The h a i r f o l l i c l e of t he newborn p r e p u t i a l . g l a n d ( a ) . I t s o u t e r r o o t s hea th c e l l s ( b l a c k a r r ow) a re c o n t i n u o u s w i t h the c u b o i d a l - s h a p e d p e r i p h e r a l c e l l s o f the g l a n d . 228 X. F i g . 12. L o n g i t u d i n a l s e c t i o n of the h a i r f o l l i c l e of t he newborn p r e p u t i a l g l a n d , showing i t s h a i r s h a f t (a) emerg ing i n t o the d i s t a l end of the e x c r e t o r y d u c t ( b ) . .228 X. - 8 5 -PLATE 4 - The Development of the P r e p u t i a l G l and P i g . 13« The p r e p u t i a l g l a n d of t he male mouse, a t 1 week of age. 91 X . F i g . 14. The p r e p u t i a l g l a n d o f the male mouse, a t 2 weeks of age. The a c i n i a re densely-packed t o g e t h e r , and more u n i f o r m l y shaped a t t h i s s tage o f deve lopment . 91 X. F i g . 15. The main e x c r e t o r y duc t o f the 1 week o l d p r e p u t i a l g l a n d ( m a l e ) , showing i t s k e r a t i n i z e d s t r a t i f i e d squampus e p i t h e l i u m . 106 X. 1 4 -86-PLATE 5 - The Aging of the Preputial Gland Fig. 16. The preputial gland of the male at 8 weeks. The duct system has become more extensive by ductular transformation. 13 X. Fig. 17. The preputial glancl of the female, at 8 weeks. 37 X. Fig. 18. The preputial gland of the male at 5 months, showing the s t r i k i n g decrease i n acinar tissue, accompanied by a compensatory increase in the duct system. 11 X. Fig. 19. The preputial gland of the female at 5 months. Note the decrease i n acinar tissue, the abundant stroma. 37 X. -87-PLATE 6 - The A g i n g of the P r e p u t i a l G land F i g . 20. The p r e p u t i a l g l a n d of the m a l e , a t 11 months. The g l a n d i s c y s t i c . 13 X. F i g . 21 . The p r e p u t i a l g l a n d of the f e m a l e , a t 11 months. The parenchyma i s much reduced and the lumen of the c e n t r a l duc t has i n c r e a s e d c o n s i d e r a b l y . 37 X. F i g . 22. D e t a i l o f the 11 mpnth o l d c y s t i c p r e p u t i a l g l a n d of the ma l e . The c e n t r a l duc t i s f i l l e d w i t h a subs tance r i c h i n l y m p h o c y t e s , po l ymo rphonuc l ea r n e u t r o p h i l e s and desquamated e p i t h e l i a l c e l l s . On ly a few i s o l a t e d a c i n i (a) rema in a t the p e r i p h e r y of the g l a n d . 37 X . - 8 8 -PLATE 7 - The E x p e r i m e n t a l G lands of Group I (Ma l e s ) P i g . 23. The p r e p u t i a l g l a nd of the male mouse a t 7 weeks, showing the no rma l , f u l l y d e v e l o p e d , compound a l v e o l a r p a t t e r n . 12 X. F i g . 24. The p r e p u t i a l g l a n d of the 10 week c a s t r a t e d ma le . The g l a n d i s a t r o p h i c . Compare w i t h f i g u r e 23, 12 X. F i g . 25. The p r e p u t i a l g l a n d of the 10 week c a s t r a t e d ma le , a f t e r the d a i l y a d m i n i s t r a t i o n of e s t r a d i o l benzoa te f o r 3 weeks. The g l a n d shows a s i m i l a r degree of a t r o p h y as does the c o n t r o l g l a nd i n f i g u r e 24. 12 X. F i g . 26, The p r e p u t i a l g l a n d of the 10 week c a s t r a t e d ma le , a f t e r the d a i l y a d m i n i s t r a t i o n of t e s t o s t e r o n e p r o p i o n a t e f o r 3 weeks. The t e s t o s t e r o n e t r e a t m e n t has c o u n t e r a c t e d the p o s t - c a s t r a t i o n a t r o p h y of the g l a n d , t h e r e b y m a i n t a i n i n g the a d u l t s t r u c t u r a l p a t t e r n . Compare w i t h f i g u r e s 23 and 24. 12 X. F i g . 27. The p r e p u t i a l g l a n d of a n o t h e r 10 week c a s t r a t e d ma le , a f t e r the t e s t o s t e r o n e t r e a t m e n t , showing the " e x h a u s t i o n a t r o p h y " w h i c h i s the r e s u l t of hormona l o v e r s t i m u l a t i o n . 12 X. - 8 9 -PLATE 8 - The E x p e r i m e n t a l G lands of Group I (Ma l e s ) F i g . 28. D e t a i l of the p r e p u t i a l g l a nd of the 10 week c a s t r a t e . Note t he abundant f i b r o u s i n t e r -a c i n a r and s u b e p i t h e l i a l s t r oma , and t h e s m a l l , u nde rdeve l oped a c i n i . Compare w i t h f i g u r e 6. 146 X. F i g . 29. D e t a i l o f the p r e p u t i a l g l a n d i n f i g u r e 27 -the 10 week c a s t r a t e d g l a n d o v e r s t i m u l a t e d by-p r o l o n g e d t e s t o s t e r o n e t r e a t m e n t s . Lymphocytes and p o l y m o r p h o n u c l e a r n e u t r o p h i l s (a ) i n vade the d u c t s , a c i n i and s t roma of the g l a n d . 146 X. F i g . 30. D e t a i l o f the e s t r a d i o l - t r e a t e d , 10 week c a s t r a t e d p r e p u t i a l g l a n d ( m a l e ) , showing the d i r e c t e f f e c t s of the e s t r o g e n i c hormone -the t h i c k e n e d s t r a t i f i e d e p i t h e l i u m of the c e n t r a l d u c t , and the desquamated e p i t h e l i a l c e l l s i n the d u c t . 146 X. - 9 0 -PLATE 9 - The E x p e r i m e n t a l G lands of Group I (Females) F i g . 31 . The p r e p u t i a l g l a n d o f the fema le mouse, a t 7 weeks. 41 X. F i g . 32. The p r e p u t i a l g l a n d o f the 10 week c a s t r a t e d f e m a l e . The g l a n d i s no t a t r o p h i c l i k e t h a t of the c a s t r a t e d male ( f i g u r e 2 4 ) . Only-a g i n g e f f e c t s are ob se rved - the dec rea se i n the amount of parenchyma, the i n c r e a s e i n the lumen o f the c e n t r a l d u c t , f o r example 41 X . F i g . 33. The p r e p u t i a l g l a n d of the 10 week c a s t r a t e d f e m a l e , a f t e r p r o l o n g e d e s t r a d i o l benzoate t r e a t m e n t s . 41 X. F i g . 34. D e t a i l o f f i g u r e 33, showing the d i r e c t e f f e c t s of the e s t r o g e n i c hormone. 122 X. -91-PLATE 10 - The E x p e r i m e n t a l G lands of Group I ( Fema le s ) F i g . 35. The p r e p u t i a l g l a n d of the 10 week c a s t r a t e d fema le a f t e r p r o l o n g e d t e s t o s t e r o n e t r e a t m e n t , showing the s t i m u l a t o r y e f f e c t of the hormone, as m a n i f e s t by marked h y p e r p l a s i a of t he a c i n a r c e l l s , h y p e r t r o p h y of the a c i n i and i n c r e a s e d d u c t u l a r t r a n s f o r m a t i o n . Compare w i t h f i g u r e 32. 41 X. F i g . 36. D e t a i l of f i g u r e 35 - t he s t i m u l a t e d f ema le g l a n d . Note the h y p e r t r o p h i e d a c i n i , t he absence of abundant i n t e r a c i n a r s t r oma , a l l of w h i c h g i v e the g l a n d a m a l e - l i k e s t r u c t u r a l p a t t e r n . Compare w i t h f i g u r e s 6 and 42. 122 X. - 9 2 -PLATE 11 - The E x p e r i m e n t a l G lands of Group I i - I n t a c t A d u l t M i ce (Ma le s ) F i g . 37. The p r e p u t i a l g l a n d of the 10 week i n t a c t ma,le mouse. Compare w i t h t he 10 week c a s t r a t e g l a nd i n f i g u r e 24. 12 X. F i g . 38. The p r e p u t i a l g l a n d of the 10 week i n t a c t ma le , a f t e r p r o l o n g e d t e s t o s t e r o n e t r e a t m e n t , showing the s t i m u l a t o r y e f f e c t of t he hormone as m a n i f e s t by i n c r e a s e d d u c t u l a r t r a n s f o r m -a t i o n . Compare w i t h f i g u r e 37. 12 X. F i g . 39. The p r e p u t i a l g l a n d of the 10 week i n t a c t ma le , a f t e r a p r o l o n g e d e s t r a d i o l t r e a t m e n t , showing the s t r i k i n g a t r o p h y of the g l a n d . Compare w i t h f i g u r e 37. 12 X. F i g . 40. D e t a i l of f i g u r e 39. Note the s m a l l - s i z e a c i n i and the r a t h e r more abundant f i b r o u s i n t e r a c i n a r and s u b e p i t h e l i a l s t r oma . 122 X. PLATE 12 - The E x p e r i m e n t a l G lands of Group I i - I n t a c t A d u l t M i ce (Females ) P i g . 41 . The p r e p u t i a l g l and o f the i n t a c t f ema le a t 10 weeks. 41 X. F i g . 42. D e t a i l o f f i g u r e 41 . Note t he t y p i c a l a g i n g e f f e c t s wh i ch o ccu r i n the norma l f ema le g l a nd - the d e c r e a s e i n the amount of parenchyma, the abundant s t r oma , the i n v a s i o n of some a c i n i by c o n n e c t i v e t i s s u e e lement s ( a ) . 122 X. F i g . 43 . The p r e p u t i a l g l a n d of the 10 week i n t a c t f e m a l e , a f t e r p r o l o n g e d t e s t o s t e r o n e t r e a t -ment, showing the s t i m u l a t o r y e f f e c t s of t he hormone - the h y p e r p l a s i a of t he a c i n a r c e l l s , t he h y p e r t r o p h y of the a c i n i , and the i n c r e a s e d d u c t u l a r t r a n s f o r m a t i o n . Compare w i t h f i g u r e 41. 41 X. F i g . 44. The p r e p u t i a l g l a n d of the 10 week i n t a c t f e m a l e , a f t e r p r o l o n g e d e s t r a d i o l t r e a t m e n t , showing the d i r e c t e f f e c t s of the hormone -the m e t a p l a s i a of t he squamous e p i t h e l i u m of the d u c t s and the de squamat ion of e p i t h e l i a l c e l l s i n t o the d u c t s . 41 X. - 9 4 -PLATE 13 - The E x p e r i m e n t a l G lands of Group I I - W e a n l i n g M i c e (Ma le s ) F i g . 45. The p r e p u t i a l g l a n d o f the 3 week male mouse wh i ch has no t y e t a t t a i n e d the a d u l t s t r u c t u r a l p a t t e r n . 41 X. F i g . 46. The p r e p u t i a l g l a nd of the 6 week no rma l male mouse w h i c h e x h i b i t s t he a d u l t s t r u c t u r a l p a t t e r n . Compare w i t h f i g u r e 45. 41 X. F i g . 47. The p r e p u t i a l g l and of the 6 week male a f t e r p r o l o n g e d t e s t o s t e r o n e t r e a t m e n t . There i s some i n d i c a t i o n of i n c r e a s e d s e c r e t o r y a c t i v i t y as m a n i f e s t by h y p e r p l a s i a of the a c i n a r c e l l s and by h y p e r t r o p h y of the a c i n i . Compare w i t h f i g u r e 46. 41 X. F i g . 48. The p r e p u t i a l g l a n d of the 6 week male a f t e r p r o l o n g e d e s t r a d i o l t r e a t m e n t , showing the i n h i b i t i o n of i t s no rma l g rowth by the hormone. No te the s m a l l - s i z e a c i n i , the more abundant s t r oma , the u n d e r d e v e l o p e d d u c t s y s tem. Compare w i t h f i g u r e 46. 41 X. - 9 5 -PLATE 14 T The E x p e r i m e n t a l G lands of Group I I - W e a n l i n g M i c e (Fema le s ) F i g . 49. The p r e p u t i a l g l a n d of the f ema le mouse a t 3 weeks. 41 X. F i g . 50. The p r e p u t i a l g l a n d of the f ema le mouse a t 6 weeks. 41 X, . F i g . 51. The p r e p u t i a l g l a n d of t he f ema le mouse a t 6 weeks a f t e r p r o l o n g e d t e s t o s t e r o n e gtim-n u l a t i o n w h i c h i s m a n i f e s t by h y p e r p l a s i a of t he a c i n a r c e l l s , h y p e r t r o p h y of the a c i n i and by i n c r e a s e d d u c t u l a r t r a n s f o r m a t i o n . Compare w i t h f i g u r e 50. 41 X. F i g . 52. The p r e p u t i a l g l a n d of the fema le mouse a t 6 weeks, a f t e r p r o l o n g e d e s t r a d i o l t r e a t m e n t . Compare w i t h f i g u r e 50 and no te the s m a l l amount of parenchyma composed of s m a l l a c i n i , t he v e r y abundant s t r oma , t he m e t a p l a s i a of the d u c t u l a r e p i t h e l i u m , and the desquamat ion of e p i t h e l i a l c e l l s i n t o the c e n t r a l d u c t . 41 X. - 9 6 -PLATE 15 - The E x p e r i m e n t a l G lands o f Group I I I - Newborn M i c e F i g . 53. The p r e p u t i a l g l a n d of the male mouse a t 3 weeks, a f t e r p r o l o n g e d t e s t o s t e r o n e s t i m -u l a t i o n , m a n i f e s t by h y p e r p l a s i a o f t he a c i n a r c e l l s and i n c r e a s e d d u c t u l a r t r a n s -f o r m a t i o n . Compare w i t h the norma l 3 week male g l a n d i n f i g u r e 45. 41 X. F i g . 54. The p r e p u t i a l g l a n d of t h e - f e m a l e mouse a t 3 weeks, a f t e r p r o l o n g e d t e s t o s t e r o n e t r e a t m e n t , showing the s t i m u l a t o r y e f f e c t s of the hormone. Compare w i t h the normal 3 week fema le g l a n d i n f i g u r e 49. 41 X. F i g . 55. The p r e p u t i a l g l a n d of the male mouse a t 3 weeks, a f t e r p r o l o n g e d e s t r a d i o l t r e a t m e n t , Compare w i t h f i g u r e 45. 41 X. F i g . 56. The p r e p u t i a l g l a n d of the fema le mouse a t 3 weeks a f t e r p r o l o n g e d e s t r a d i o l t r e a t -ment. Compare w i t h f i g u r e 49. 41 X. -97-PLATE 16 - The Experimental Glands of Group III - Newborn Mice Fig. 57. De t a i l of the normal 3 week preputial gland (male). 122 X. Fig. 58. De t a i l of figure 53, the 3 week testosterone-treated preputial gland (male). Note the hypertrophy of the a c i n i . Compare with figure 57. 122 X. Fig. 59- Detail of figure 56, the 3 week e s t r a d i o l -treated preputial gland (female). Note i t s resemblance, generally, with the normal 3 week gland i n figure 57. 122 X. -98-PLATE 17 - H i s t o c h e m i s t r y F i g . 60. Sudan b l a c k B s t a i n i n g , d e m o n s t r a t i n g the d e p o s i t i o n of l i p i d d r o p l e t s i n the a c i n a r c e l l s of the mouse p r e p u t i a l g l and ( a d u l t f e m a l e ) . The l a r g e s t d r o p l e t s c o a l e s c e i n the d e g e n e r a t i n g c e l l s o f t he h y p e r t r o p h i e d a c i n i ( a ) . 304 X. F i g . 61. McManus' Sudan b l a c k B method. The l i p i d d r o p l e t s are me re l y h e a v i l y o u t l i n e d by the s t a i n . 304 X. F i g . 62. D i f f e r e n t i a l s t a i n i n g t e c h n i q u e of Beaver ( i 9 6 0 ) , showing the d e p o s i t i o n of o i l r e d 0 d r o p l e t s t h roughou t the a c i n a r c e l l s of the mouse p r e p u t i a l g l and ( a d u l t f e m a l e ) . A g a i n the l a r g e s t d r o p l e t s c o a l e s c e i n the d e g e n e r a t i n g c e l l s of t he h y p e r t r o p h i e d a c i n i ( a ) . 304 X. PLATE 18 - H i s t o c h e m i s t r y P i g . 63. B i e b r i c h S c a r l e t method of S p i c e r and L i l l i e , at pH 4.95, demonstrating the p e r i n u c l e a r proteinaceous granules i n the a c i n a r c e l l s of the r a t p r e p u t i a l gland-(female). 304 X. F i g , 64. M a l l o r y ' s phosphot-ungstic a c i d hematoxylin method, demonstrating the p r o t e i n granules i n the a c i p a r c e l l s of the r a t p r e p u t i a l gland (female). 304 X. F i g . 65. Heidenhain's i r o n hematoxylin method, demonstrating the p e r i n u c l e a r , proteinaceous granules i n the a c i n a r c e l l s of the r a t pre-p u t i a l gland (female). Note t h a t the granules arq most numerous and l a r g e i n s i z e i n the a c i n a r c e l l s approaching degeneration- 304 X. F i g . 66. Heidenhain's i r o n hematoxylin method a p p l i e d to the mouse p r e p u t i a l gland (male). Note the complete absence of granules. Compare with f i g u r e 66. 304 X. -100-PLATE 19 - H i s t o c h e m i s t r y F i g . 67. D i f f e r e n t i a l s t a i n i n g t e c h n i q u e of Beaver ( i 960 ) showing the d e p o s i t i o n of o i l :red 0 d r o p l e t s ( red ) t h roughou t the a c i n a r c e l l s o f the mouse p r e p u t i a l g l a n d ( a d u l t , f e m a l e ) . 80 X. F i g . 68. D i f f e r e n t i a l s t a i n i n g t e c h n i q u e o f Beaver ( i 9 6 0 ) , showing the randprn d e p o s i t i o n o f o i l r e d 0 d r o p l e t s ( red ) and dense p r o t e i n g r a n u l e s w h i c h are m a i n l y p e r i n u c l e a r i n p o s i t i o n ( b l a c k arrow) i n the a c i n a r c e l l s of the r a t p r e p u t i a l g l a nd ( f e m a l e ) . The g r a n u l e s c o a l e s c e i n the duc t s of the g l a n d ( a ) . Compare w i t h f i g u r e 67 i n wh i ch the p r o t e i n g r a n u l e s are a b s e n t . 108 X. . F i g . 69. O i l r e d 0 s t a i n i n g f o l l o w e d by H e i d e n h a i n ' s i r o n h e m a t o x y l i n method, d e m o n s t r a t i n g the d i s t r i b u t i o n of dense p r o t e i n g r a n u l e s ( b l ue -b l a c k ) and o i l r e d 0 d r o p l e t s s i m u l t a n e o u s l y i n the a c i n i of the r a t p r e p u t i a l g l a n d (ma l e ) . 136 X. -101-A CKN 0WLE D GEMEN T S S i n c e r e thanks are ex tended t o Dr . J . M. T a y l o r f o r her a d v i c e and a s s i s t a n c e i n the g e n e r a l s u p e r v i s i o n of the s t u d y . I a l s o w i s h t o thank Dr . P. F o r d and Dr . A. M. Pe r k s who p r o v i d e d h e l p f u l c r i t i c i s m and a d v i c e . I am g r a t e f u l t o Doro thy K i e s e r f o r her t r a n s -l a t i o n of s e v e r a l German a r t i c l e s , to Daphne Hards f o r he r v a l u a b l e t e c h n i c a l s k i l l w i t h the h i s t o c h e m i c a l p r o c e d u r e s , and t o a l l the o t h e r peop le who h e l p e d me. F i n a l l y , I w i s h to e s p e c i a l l y thank Den i s f o r h i s encouragement and h e l p t h roughou t the cour se of the s t u d y . - 1 0 2 -LITERATURE CITED A r a i , Y. 1968. M e t a p l a s i a i n male r a t r e p r o d u c t i v e a c c e s s o r y g l and s i nduced by n e o n a t a l e s t r o g e n t r e a t m e n t . E x p e r e n t i a 24: 180-18,1. B a r n e s , H.H.F. and G. I .M. Swyer. 1951. E f f e c t of g rowth hormone and of c h o r i o n i c g o n a d o t r o p h i n on p r e p u t i a l g l and s of the fema le r a t . B r i t . Med. J . 2: 207-208, Beave r , D.L. 1959. Der E i n f l u s s v e r s c h i e d e n e r Pix ierungs-^-m i t t e l auf das S t r t i k t u r b i l d der P r a p u t i a l d r u s e n der R a t t e . Z t s c h r . Z e l l f o r s c h . m i k r . Anat.- 51; 88-96 Beave r , D.L. 1960. The r e - e v a l u a t i o n of the r a t p r e p u t i a l g l a n d as a " d i c r i n e " o rgan f rom the s t a n d p o i n t of i t s morpho logy , h i s t o c h e m i s t r y , and p h y s i o l o g y . J . Exp . Z o o l . 143: 15,3-173. B r on son , F .H. 1966. A sex a t t r a c t a n t f u n c t i o n f o r mouse p r e p u t i a l g l a n d s . Amer. Z o o l . 6: 535. A b s t r a c t . B r on son , F . H . , C-P. Dagg and G.D. S n e l l . 1966. R e p r o d u c t i o n . I n B i o l o g y of the L a b o r a t o r y Mouse, (E. G reen , e d . ) , 2nd e d i t i o n , The J a c k s o n L a b o r a t o r y . B r o o k s , S .C. , J . J . L a l i c h , C.A. Baumann. 1956. 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