UBC Theses and Dissertations

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UBC Theses and Dissertations

Degeneration of the germinal epithelium in the mouse and rat testis with respect to the seminiferous… Langford, George Albert 1967

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DEGENERATION OF THE GERMINAL EPITHELIUM IN THE MOUSE AND RAT TESTIS WITH RESPECT TO THE SEMINIFEROUS CYCLE FOLLOWING LIGATION OF THE VASA EFFERENTIA by  B.Sc,  GEORGE ALBERT LANGFORD U n i v e r s i t y o f B r i t i s h Columbia, 1964  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e Department of ZOOLOGY  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1967  In presenting t h i s thesis in p a r t i a l  f u l f i l m e n t of the requirements  for an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and Study.  I further agree that permission for extensive copying of  this  thesis for s c h o l a r l y purposes may be granted by the Head of my Department or by h.i.<s representatives.  It  is understood that copying  or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission.  Department of  ZOOLOGY  The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date  NOVEMBER 6, 1967  ABSTRACT  The manner i n w h i c h t h e c y c l e o f t h e g e r m i n a l e p i t h e l i u m i s a f f e c t e d d u r i n g p r o g r e s s i v e a t r o p h y o f semi n i f e r o u s t u b u l e s , and t h e form o f c e l l u l a r  degeneration  t h a t o c c u r s a t each stage o f t h e c y c l e has been s t u d i e d i n a d u l t r a t and mouse t e s t i s f o l l o w i n g u n i l a t e r a l the vasa  l i g a t i o n of  efferentia. F o l l o w i n g t h e l i g a t i o n , w h i c h was p l a c e d as c l o s e  t o t h e t e s t i s as p o s s i b l e , groups o f animals were k i l l e d a t p o s t o p e r a t i v e p e r i o d s r a n g i n g from 1 t o 10 days f o r r a t s , and  16 h o u r s t o 7 days f o r m i c e .  The stages o f t h e c y c l e  were i d e n t i f i e d by t h e p e r i o d i c a c i d - S c h i f f - h a e m a t o x y l i n technique  u s i n g t h e c l a s s i f i c a t i o n o f Leblond  (1952) and Oakberg The  and Clermont  (1956a).  t i m i n g o f t h e c y c l e d i d n o t appear t o be d i s -  r u p t e d by t h e d e g e n e r a t i v e  changes t h a t r e s u l t e d :  at a l l  p o s t o p e r a t i v e p e r i o d s , t h e v i a b l e germ c e l l s were a s s o c i a t e d w i t h t h e same s t a g e s o f t h e c y c l e as t h e y a r e i n t h e normal t e s t i s .  The f r e q u e n c y  w i t h which each stage o f t h e  c y c l e o c c u r r e d was measured i n t h e l i g a t e d  t e s t i s of the  mouse and found t o be unchanged from t h a t e s t a b l i s h e d i n the c o n t r o l t e s t i s . ii  The a t r o p h i c r e a c t i o n s a t each s t a g e o f t h e c y c l e f o l l o w e d a c o n s i s t e n t and w e l l - d e f i n e d sequence o f e v e n t s which have been d e s c r i b e d i n t h e form o f a g e n e r a l scheme. The proposed scheme has n o t p r e v i o u s l y been d e s c r i b e d i n t h e l i t e r a t u r e , and c o n s i s t s o f 4 main s t e p s i n t u b u l e degeneration. (1)  D u r i n g s t a g e s 1 - IX o f t h e c y c l e t h e s e s t e p s a r e :  the "spermatid s l o u g h i n g " stage,  l e a t e d body" s t a g e , and  (4)  (3)  (2)  the "multinuc-  the " S e r t o l i c e l l cytoplasm"  the " S e r t o l i c e l l sloughing" stage.  During  stage, stages  X - X l l o f t h e c y c l e i n t h e mouse ( s t a g e s X - XIV i n t h e r a t ) , t h e f i r s t 2 o f t h e 4 d e g e n e r a t i v e s t e p s d i f f e r and have been termed: (2)  (1)  t h e "spermatocyte Degeneration  t h e " c e l l u l a r s l o u g h i n g " s t a g e , and ghost c e l l "  stage.  of seminiferous tubules a f f e c t e d  S e r t o l i c e l l s i n a d e f i n i t e manner:  the S e r t o l i c e l l  nuclei  i n c r e a s e d i n number, f r e q u e n t l y m i g r a t e d away from t h e b a s e ment membrane and were o f t e n sloughed These changes i n t h e b e h a v i o u r  i n t o t h e t u b u l e lumen.  of S e r t o l i c e l l s d u r i n g tub-  u l a r atrophy are d i s c u s s e d .  iii  TABLE OF CONTENTS  Abstract  i i  Table of Contents  iv  L i s t of Tables  vi  Acknowledgements  v i i  INTRODUCTION  1  MATERIALS AND METHODS  6  1.  L i g a t i o n o f Vasa E f f e r e n t i a i n R a t s  7  11.  L i g a t i o n o f Vasa E f f e r e n t i a i n M i c e  8  Electron Microscopy  9  111. RESULTS  13 1.  L i g a t i o n o f t h e Vasa E f f e r e n t i a i n Rats  13  11.  L i g a t i o n o f t h e Vasa E f f e r e n t i a i n M i c e  13  A. B. C. D.  Changes i n t h e C y c l e o f t h e S e m i n i f erous E p i t h e l i u m  13  Changes i n t h e S i z e o f S e m i n i f e r o u s Tubules  15  Changes i n t h e Number o f S e r t o l i Cell Nuclei  17  H i s t o l o g i c a l Changes i n t h e Epididymis  18  iv  TABLE OF CONTENTS (Con't)  E.  F.  H i s t o l o g i c a l Changes i n t h e G e r m i n a l Epithelium  18  1.  D e g e n e r a t i v e Steps i n Stages X - X l l of the Cycle  22  2.  D e g e n e r a t i v e Steps i n Stages 1 - IX o f t h e C y c l e  23  3.  The F a t e o f M u l t i n u c l e a t e d B o d i e s  26  4.  The F a t e o f R e s i d u a l B o d i e s  28  A t r o p h i c R e a c t i o n s i n Normal T i s s u e  29  DISCUSSION  30  SUMMARY  38  LITERATURE CITED  41  KEY TO ABBREVIATIONS  46  PLATES  V  LIST OF TABLES  T a b l e 1.  The c y c l e of t h e s e m i n i f e r o u s e p i t h e l i u m i n the r a t .  11  T a b l e 2.  The c y c l e o f t h e s e m i n i f e r o u s e p i t h e l i u m i n t h e mouse.  12  T a b l e 3.  P e r c e n t a g e w i t h which each s t a g e o f t h e seminiferous c y c l e occurs f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a i n the mouse.  14  T a b l e 4.  Mean d i a m e t e r o f 20 s e m i n i f e r o u s t u b u l e s from each p o s t o p e r a t i v e s t a g e i n m i c e .  16  T a b l e 5.  Mean S e r t o l i c e l l c o u n t s from 20 semini f e r o u s t u b u l e c r o s s s e c t i o n s a t each p o s t o p e r a t i v e stage i n mice.  17  T a b l e 6.  The h i s t o l o g i c a l changes i n t h e g e r m i n a l epithelium f o l l o w i n g l i g a t i o n of the v a s a e f f e r e n t i a i n mice.  20  vi  ACKNOWLEDGEMENTS  I would l i k e t o e x p r e s s my s i n c e r e thanks t o Dr. A.B. A c t o n f o r h i s c o n t i n u e d s u p p o r t and encouragement, and f o r a l l o w i n g me t o chose t h e c u r r e n t r e s e a r c h problem. Thanks a r e a l s o due t o Dr. C.V. Finnegan & Dr. P. F o r d f o r their criticism  of the manuscript.  Finally,  I would  like  t o acknowledge t h e i n t e r e s t o f D r . J.A. B i r k b e c k a t t h e Vancouver G e n e r a l H o s p i t a l .  vii  INTRODUCTION  Since the d e s c r i p t i o n of the c y c l e of the s e m i n i f e r o u s e p i t h e l i u m i n the t e s t e s of c e r t a i n mammals i n terms of d e v e l o p mental r e l a t i o n s h i p s of a l l germ c e l l s 1952), i t i s now  Clermont,  p o s s i b l e t o study s y s t e m a t i c a l l y the c e l l u l a r  events c o m p r i s i n g spermatogenesis c e l l s , type A spermatogonia, matozoa.  (Leblond and  - from the e a r l i e s t germ,  t o m o r p h o l o g i c a l l y mature s p e r -  P r e v i o u s l y , no adequate r e l i a b l e method had been  d e v i s e d f o r c l a s s i f y i n g the d i f f e r e n t c e l l u l a r of germ c e l l s  associations  (Roosen - Runge and G i e s e l , 1950).  r e p o r t by Leblond and Clermont  The  (1952), s t i m u l a t e d a l a r g e  number of d e t a i l e d i n v e s t i g a t i o n s of the c y c l e of the i f e r o u s e p i t h e l i u m i n v a r i o u s animals Clermont  and Leblond, 1955,  Clermont  and Perey, 1957;  Oakberg, 1955  a,b,  1956  1959;  semin-  (Clermont, 1954,  Daoust and Clermont,  Perey, Clermont  a,b;  initial  and Leblond,  Sapsford, 1962,  1963; 1955; 1961;  a,b), but these  r e p o r t s a l l f o c u s a t t e n t i o n on the c y c l e as a p p l i e d t o s p e r matogenesis  i n the normal  testis*  The exact manner, however, i n which the c y c l e i s a f f e c t e d d u r i n g p r o g r e s s i v e t e s t i c u l a r atrophy, and the type of c e l l u l a r d e g e n e r a t i o n t h a t occurs a t v a r i o u s stages of the c y c l e , has not been w e l l d e f i n e d .  The purpose of t h i s  thesis  i s t o c l a r i f y these two problems i n the mouse and i n the r a t .  2  The c l a s s i f i c a t i o n of the c y c l e of the s e m i n i f e r o u s e p i t h e l i u m i s based on w e l l d e f i n e d steps i n the development of  spermatids t o m o r p h o l o g i c a l l y mature spermatozoa.  Differ-  ences i n the acrosomic and head cap r e g i o n s of spermatids, as seen a f t e r p e r i o d i c a c i d - S c h i f f s t a i n i n g , have made i t p o s s i b l e to  d e s c r i b e 19 steps i n the r a t (Leblond and Clermont,  and 16 s t e p s i n the mouse (Oakberg, of  1956a).  The f i r s t  spermatid development i n the r a t , and the f i r s t  1952), 14 steps  12 steps i n  the mouse, d e f i n e the stages of the s e m i n i f e r o u s c y c l e i n each animal.  A b r i e f e x p l a n a t i o n of the c y c l e w i t h r e s p e c t t o the  spermatids i s presented w i t h p l a t e  1.  A c c o r d i n g t o Leblond and Clermont, spermatogenesis  extends over 4 complete  the p r o c e s s of  c y c l e s of the s e m i n i f -  erous e p i t h e l i u m , b e g i n n i n g i n stage IX w i t h mitoses i n type A spermatogonia,  and ending w i t h the r e l e a s e of spermatozoa i n  stage V l l l of the c y c l e . mine how  I t i s t h e r e f o r e important t o d e t e r -  the c y c l e i s a f f e c t e d d u r i n g p r o g r e s s i v e t e s t i c u l a r  atrophy, s i n c e the f a c t o r s t h a t are r e s p o n s i b l e f o r the d e v e l opmental r e l a t i o n s h i p s of a l l germ c e l l s i n the c y c l e are a l s o the f a c t o r s which determine the r a t e of In hypophysectomized  spermatogenesis.  r a t s , Clermont  and  Morgentaler  (1955) observed t h a t the c y c l e of the s e m i n i f e r o u s e p i t h e l i u m was  not a f f e c t e d , although the numbers of  spermatogonia,  3  spermatocytes and spermatids were g r e a t l y reduced.  This  v e r i f i e d by the r a d i o a u t o g r a p h i c r e s u l t s of Clermont Harvey  was  and  (1965), which showed t h a t the r e l a t i v e d u r a t i o n s of  each stage of the c y c l e i n t e s t e s from hypophysectomized  rats  were e s s e n t i a l l y unchanged from the normal c o n t r o l t e s t e s . R e c e n t l y , Chowdhury and S t e i n b e r g e r (1964) r e p o r t e d t h a t the frequency of occurrence of each stage i n the  spermatogenic  c y c l e remains r e l a t i v e l y unchanged when r a t t e s t e s are exposed o t o 43 C.  At t h i s temperature, however, o n l y a p a r t i a l degen-  e r a t i o n of the g e r m i n a l e p i t h e l i u m o c c u r s .  Whether the c y c l e  i s a f f e c t e d d u r i n g more severe c o n d i t i o n s of atrophy has not been determined. In order t o study the exact manner i n which the c y c l e of the s e m i n i f e r o u s e p i t h e l i u m i s a f f e c t e d d u r i n g a p r o g r e s s i v e d e g e n e r a t i o n of the g e r m i n a l e p i t h e l i u m , a method of i n d u c i n g t e s t i c u l a r atrophy was  adopted which i s not depen-  dent on the heat s e n s i t i v i t y of the d i f f e r e n t germ c e l l s , nor on the i n f l u e n c e of s p e c i f i c p i t u i t a r y hormones. s e l e c t e d was  l i g a t i o n of the v a s a e f f e r e n t i a .  The method  The e f f e c t of  t h i s o p e r a t i o n on the t e s t i s i n mice has not p r e v i o u s l y been r e p o r t e d , but e a r l i e r s t u d i e s of l i g a t i o n o f the v a s a e f f e r e n t i a i n rats  (Van Wagenen, 1924,  1925,  1926;  Smith,  1962),  i n d i c a t e d t h a t a r a p i d and complete d e g e n e r a t i o n of the g e r m i n a l  4  epithelium s i d e r how describe various  i s produced.  These r e p o r t s , however, do not  the spermatogenic  con-  c y c l e i s a f f e c t e d , nor do they  the type of c e l l u l a r d e g e n e r a t i o n t h a t occurs at stages of the c y c l e i n d e t a i l .  s e m i n i f e r o u s t u b u l e undergoing  An example of a  severe atrophy  following  l i g a t i o n of the vasa e f f e r e n t i a i s shown i n f i g u r e 1.  F i g . 1. S e m i n i f e r o u s t u b u l e o f a r a t f o l l o w i n g l i g a t i o n of t h e v a s a e f f e r e n t i a f o r 6 d a y s . Extreme a t r o p h y d u r i n g t h e e a r l y s t a g e s of t h e c y c l e o f t h e s e m i n i f e r o u s e p i t h e l i u m has r e s u l t e d . Numerous m u l t i n u c l e a t e d b o d i e s (MNB) o f immature s p e r m a t i d s are found i n t h e g e r m i n a l e p i t h e l i u m i n v a r i o u s s t a t e s of d e g e n e r a t i o n ; one m u l t i n u c l e a t e d body a t t h e lumen of t h e t u b u l e (L) i s about t o be e x f o l i a t e d . Many S e r t o l i c e l l nuclei (SC) are d i s p l a c e d away from t h e basement membrane (BM) and occupy a p o s i t i o n a d j a c e n t t o m u l t i n u c l e a t e d b o d i e s , n e c r o t i c pachytene spermatocytes (PC), and a t t h e edges of v a c u o l e s (V). Spermatogonia (SG) s t i l l retain t h e i r normal n u c l e a r morphology and p o s i t i o n a d j a c e n t t o t h e basement membrane. Iron haematoxylin. X 1350.  5  6  MATERIALS AND METHODS  The o r g a n i z a t i o n of the c y c l e of the s e m i n i f e r o u s e p i t h e l i u m i n the r a t (Leblond and Clermont, 1952), and i n the  mouse (Oakberg, 1956  a) has been reproduced i n t a b l e s  1,  and 2, pages 11 and 12, i n order t o p r o v i d e a r e f e r e n c e f o r the  e x t e n t of development  o f the d i f f e r e n t germ c e l l s con-  s t i t u t i n g each stage of the c y c l e .  The term  "cellular  a s s o c i a t i o n " i s synonymous w i t h stage - w i t h r e s p e c t t o the c y c l e , and i n c l u d e s the type of spermatogonia,  spermatocytes  and spermatids which make up a p a r t i c u l a r r e g i o n of a seminiferous tubule.  The v e r t i c a l columns, i n d i c a t e d by Roman  numerals i n each t a b l e , r e p r e s e n t the stages o f the c y c l e . The type of spermatogonia and the developmental steps of spermatocytes i n each stage are r e p r e s e n t e d by letters;  capital  the developmental s t e p s of spermatids, i n A r a b i c  numerals;  the m i t o t i c d i v i s i o n s of spermatogonia, by  s c r i p t m;  and the m a t u r a t i o n d i v i s i o n s o f primary and  secondary spermatocytes, by s u b s c r i p t md.  sub-  The germ c e l l s  seen i n a c r o s s s e c t i o n o f any g i v e n r e g i o n of a t u b u l e i n the  r a t or mouse t e s t i s would t h e r e f o r e r e p r e s e n t o n l y one  of the c e l l u l a r a s s o c i a t i o n s o f e i t h e r t a b l e 1 or 2 respectively.  7  The a n i m a l s used i n t h e p r e s e n t e x p e r i m e n t s were healthy  s e x u a l l y mature male W i s t a r r a t s (210 - 300 g ) ,  and male Swiss mice (28 - 35 g ) , m a i n t a i n e d i n a c o n t r o l l e d environment room a t 23°C.  B u c k e r f i e l d s Crumbles and water 1  were s u p p l i e d ad l i b i t u m . The h i s t o l o g i c a l p r o c e d u r e i n v o l v e d f i x a t i o n o f t e s t e s and t h e i r a d j o i n i n g e p i d i d y m i s i n B o u i n ' s f l u i d f o r 2 d a y s , f o l l o w e d by f o u r 2-hour r i n s e s i n 70% e t h a n o l .  The  t i s s u e was d e h y d r a t e d , c l e a r e d i n benzene, and embedded i n p a r a f f i n a t 59°C,  S e r i a l s e c t i o n s 5 microns t h i c k of a l l  t e s t e s and i n t a c t e p i d i d y m i d e s were made, and mounted on glass s l i d e s .  S e c t i o n s were t h e n p r o c e s s e d u s i n g t h e p e r i o d i c  a c i d - S c h i f f - h a e m a t o x y l i n method o f L e b l o n d and (1952) f o r i d e n t i f i c a t i o n o f t h e v a r i o u s  Clermont  s t a g e s of t h e c y c l e .  An a l t e r n a t e s t a i n i n g method used f o r i d e n t i f i c a t i o n o f t h e s t a g e s o f t h e c y c l e was H e i d e n h a i n ' s i r o n h a e m a t o x y l i n f o l l o w e d by b l u e i n g i n l i t h i u m c a r b o n a t e (Clermont and P e r e y , 1957) .  1,  L i g a t i o n of Vasa E f f e r e n t i a i n R a t s The r i g h t t e s t i s o f 15 r a t s was d i s p l a c e d from t h e  scrotum t h r o u g h t h e i n g u i n a l c a n a l i n t o t h e abdominal c a v i t y  8  and g e n t l y l i f t e d o u t s i d e t h e a n i m a l t h r o u g h a s m a l l public incision.  supra-  The v a s a e f f e r e n t i a were l o c a t e d a t t h e i r  o r i g i n - an a v a s c u l a r o v a l on t h e s u r f a c e o f t h e t e s t i s near the caput e p i d i d y m i d i s . suture  The l i g a t i o n was made w i t h a f i n e  ( M e a s u r o l l s i l k 000) as c l o s e t o t h e o r i g i n ; as  p o s s i b l e without d i s r u p t i n g t h e t u n i c a albuginea.  The t e s t i s  was kept, m o i s t w i t h i s o t o n i c mammalian s a l i n e , and a t t h e completion  o f t h e o p e r a t i o n i t was r e p l a c e d a l o n g t h e i n -  g u i n a l c a n a l t o t h e scrotum. mains u n d i s t u r b e d  I n t h i s way, t h e scrotum r e -  and m a i n t a i n s e s s e n t i a l l y a normal e n v i r o n -  ment once t h e t e s t i s i s r e p l a c e d . as a c o n t r o l .  The l e f t t e s t i s was used  The r a t s were d i v i d e d i n t o groups o f 3 and  k i l l e d b y means o f e t h e r vapour a t 1, 2, 4, 6 and 10 day postoperative periods.  Subcutaneous i n j e c t i o n s o f c o l c h i c i n e  (0.2 mg/100 g body w e i g h t , d i s s o l v e d i n 0.5 m l normal s a l i n e ) were g i v e n t o each group o f a n i m a l s  6 h o u r s b e f o r e t h e y were  sacrificed.  11.  L i g a t i o n o f Vasa E f f e r e n t i a i n Mice The method used above t o l i g a t e t h e v a s a e f f e r e n t i a  i n r a t s , was used t o l i g a t e t h e v a s a e f f e r e n t i a o f t h e r i g h t t e s t i s o f 3 5 mice.  The mice were t h e n d i v i d e d i n t o groups o f  9 5 and s a c r i f i c e d a t 9 hour, 16 hour, 24 hour, 2 day, 4 day, 5 day and 7 day p o s t o p e r a t i v e p e r i o d s .  Counts o f t h e 12  d i f f e r e n t s t a g e s o f t h e c y c l e , e x p r e s s e d as p e r c e n t a g e s o f t h e t o t a l number o f s t a g e s c o u n t e d , were made b y i d e n t i f y i n g the stage of every seminiferous tubule appearing i n 4 d i f f e r ent s e c t i o n s o f t e s t i s from each o f 2, 4 and 7 day p o s t o p e r ative periods.  Measurements o f t h e mean d i a m e t e r o f 20 c r o s s  s e c t i o n s o f s e m i n i f e r o u s t u b u l e s were made a t each p o s t o p e r a t i v e p e r i o d , and from each o f t h e s e t u b u l e s measured, t h e mean number o f S e r t o l i c e l l n u c l e i p e r c r o s s s e c t i o n o f s e m i n i f e r o u s t u b u l e was d e t e r m i n e d .  111.  E l e c t r o n Microscopy S m a l l segments o f s e m i n i f e r o u s t u b u l e s about 1 -  2 mm i n l e n g t h from c o n t r o l r a t t e s t e s and r a t t e s t e s  ligated  f o r 4 and 6 day p e r i o d s were f i x e d f o r 1 hour i n 6.5% g l u t a r a l d e h y d e , phosphate b u f f e r e d t o pH 7.2 a t 4°C.  After 3 rinses  i n b u f f e r , t h e t i s s u e was p o s t f i x e d i n phosphate b u f f e r e d 1% osmium t e t r o x i d e , r i n s e d t w i c e i n b u f f e r , d e h y d r a t e d i n a graded s e r i e s o f a l c o h o l s , and embedded i n Epon 812 f o r 12 o o h o u r s a t 37 C and t h e n 24 h o u r s a t 60 C.  Thin sections of  b o t h n o r m a l and a t r o p h i c t u b u l e s were s t a i n e d w i t h u r a n y l  10  a c e t a t e f o l l o w e d by l e a d c i t r a t e and examined w i t h a H i t a c h i HS 7S e l e c t r o n m i c r o s c o p e .  O b s e r v a t i o n s were c o r r e l a t e d  w i t h t h e r e s u l t s of l i g h t m i c r o s c o p y t o a s s i s t i n t h e i n t e r p r e t a t i o n o f p r o g r e s s i v e a t r o p h y a t each s t a g e o f t h e iferous  cycle.  semin-  TABLE  1*  The C y c l e o f t h e S e m i n i f e r o u s E p i t h e l i u m i n t h e Rat  18  19  19  5  6  7  8  9  10  11  P  P  P  P  P  P  B  B/R (m)  R  R  L  L  A  A  A  A  A  A  IV  V  VI  A (m) IX  15  16  16  17  17  1  2  3  4  P  P  P  P  In  In  A  A 111  In A 1  1  11  In/B (m)  * A f t e r L e b l o n d and Clermont,  VI1  Vlll  X  12  13  14  P  P  Dip  Dia/S (md)  L  L/Z  Z  Z  A (m) Xll  A  A (m) XIV  A XI  Xlll  1952  = mitotic division  R  = r e s t i n g spermatocyte  md = m e i o t i c d i v i s i o n  L  = l e p t o t e n e spermatocyte  A  = t y p e "A"  Z  = zygotene spermatocyte  In  = t y p e " I n " spermatogonia  P  = pachytene spermatocyte  B  = t y p e "B" spermatogonia  Dip  = d i p l o t e n e spermatocyte  S  = secondary spermatocyte  Dia  = d i a k i n e s i s spermatocyte  m  spermatogonia  TABLE  2*  The C y c l e o f t h e S e m i n i f e r o u s E p i t h e l i u m i n t h e Mouse 11  111  A  A  A A (m)  A/In (m)  In  IV A In/B (m)  VI A B  A B/R (m)  V l l  Vlll  A  A  R  R/L  IX A  (m)  L  X  XI  A Z  Dip  P  P  P  P  P  P  P  P  P  1  2  3  4  5  6  7  8  9  10  13  14  14  15  15  15  16  16  A  (m)  L/Z  P  X l l  Z/P Dia/S (md)  11  12  * A f t e r Oakberg, 1956 m  == m i t o t i c d i v i s i o n  R  = r e s t i n g spermatocyte  md = m e i o t i c d i v i s i o n  L  = l e p t o t e n e spermatocyte  A  Z  = zygotene spermatocyte  I n = t y p e " I n " spermatogonia  P  = pachytene spermatocyte  B  = t y p e "B" spermatogonia  D i p = d i p l o t e n e spermatocyte  S  = secondary spermatocyte  D i a = d i a k i n e s i s spermatocyte  = t y p e "A" spermatogonia  13 RESULTS  I.  L i g a t i o n o f t h e Vasa E f f e r e n t i a i n R a t s The d e g e n e r a t i v e changes i n t h e g e r m i n a l e p i t h e l i u m  f o l l o w i n g l i g a t i o n o f t h e v a s a e f f e r e n t i a i n r a t s were e s s e n t i a l l y t h e same as t h e changes d e s c r i b e d i n mice below. Examples of r a t seminiferous t u b u l e s a t s p e c i f i c steps of atrophy 7-13)  have been used t o g e t h e r  with atrophic  (plates  seminiferous  t u b u l e s from mice ( p l a t e 10) t o i l l u s t r a t e t h e g e n e r a l s t e p s i n s e m i n i f e r o u s t u b u l e d e g e n e r a t i o n d e s c r i b e d below. The s t a g e s o f t h e c y c l e were more r e a d i l y i d e n t i f i e d by l i g h t m i c r o s c o p y by e l e c t r o n m i c r o s c o p y .  T h i s i s e x e m p l i f i e d by comparing  head cap of s p e r m a t i d s as seen by each method.  g e n e s i s as seen by t h e e l e c t r o n m i c r o s c o p e ;  II.  illus-  spermio-  f i g u r e 9, and a t  h i g h e r m a g n i f i c a t i o n , f i g u r e s 57 and 58 r e p r e s e n t t h e stage, as seen by l i g h t  the  F i g u r e 14  t r a t e s t h e head cap of a s p e r m a t i d d u r i n g s t a g e V l l l o f  than  comparative  microscopy.  L i g a t i o n o f t h e Vasa E f f e r e n t i a i n Mice A.  Changes i n t h e C y c l e o f t h e S e m i n i f e r o u s E p i t h e l i u m The f r e q u e n c y w i t h which each s t a g e o f t h e c y c l e  appears i n d e g e n e r a t i v e t e s t e s i s shown i n t a b l e 3, and  was  14  TABLE  3  P e r c e n t a g e w i t h w h i c h each s t a g e of t h e s e m i n i f e r o u s c y c l e o c c u r s f o l l o w i n g l i g a t i o n of t h e v a s a e f f e r e n t i a i n mice  Stages  Control  2 days  4 days  7 days  1  11,6  10.4  13.6  13.3  11  9.1  8.3  9.8  3.9  111  4.4  5.4  7.6  3.6  IV  8.2  6.9  4.6  2.4  V  5.1  4.3  5.8  6.3  VI  8.4  7.5  6.7  7.2  Vll  9.4  10.6  9.9  10.1  Vlll  9.8  11.1  10.6  11.2  IX  7.5  8.4  5.3  8.1  X  6.6  5.9  6.1  5.2  XI  9.4  8.2  4.6  7.9  Xll  10.5  11.3  12.0  16.2  Undetermined  0.0  1.7  3.4  4.6  Tubules counted  734  628  486  566  15 used as an i n d i c a t i o n of t h e e f f e c t s o f a t r o p h y on t h e c y c l e itself.  As a t r o p h y of t h e g e r m i n a l e p i t h e l i u m p r o g r e s s e d  from 2 days t h r o u g h 7 d a y s , i d e n t i f i c a t i o n o f each s t a g e of t h e c y c l e became i n c r e a s i n g l y d i f f i c u l t . a t i o n , and i n some t u b u l e s t h e complete  Spermatid degenerl a c k of s p e r m a t i d s ,  was e x t e n s i v e enough t o make i t n e c e s s a r y t o i n c l u d e a p e r centage o f u n c l a s s i f i e d t u b u l e s .  With few e x c e p t i o n s , t h e  r e l a t i v e f r e q u e n c y of appearance of each s t a g e o f t h e c y c l e i n a t r o p h i c t e s t e s at a l l postoperative periods analyzed n o t s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l f r e q u e n c i e s .  was  No  p a r t i c u l a r c e l l u l a r a s s o c i a t i o n was a f f e c t e d more s e r i o u s l y t h a n any o t h e r stage, o f t h e c y c l e by a p p e a r i n g a g r e a t e r o r fewer number of t i m e s t h a n i n t h e normal t e s t e s .  Severe  a t r o p h y o f s e m i n i f e r o u s t u b u l e s d i d n o t appear t o d i s r u p t t h e d e v e l o p m e n t a l r e l a t i o n s h i p s of t h e r e m a i n i n g germ c e l l s i n t h e g e r m i n a l e p i t h e l i u m . The o n l y d e v i a t i o n s from t h e c o n t r o l f r e q u e n c i e s were:  (1)  d e c r e a s e s i n s t a g e s IV and X I o f t h e  4 day p o s t l i g a t i o n p e r i o d , and s t a g e s 11 and IV o f t h e 7 day p e r i o d , and  (2)  i n c r e a s e s i n s t a g e 111 a t 4 d a y s , and  stage  X l l a t 7 days. B.  Changes i n t h e S i z e o f S e m i n i f e r o u s Tubules In c r o s s s e c t i o n s of s e m i n i f e r o u s t u b u l e s t h e r e was  an i n c r e a s e i n t h e average d i a m e t e r o f t u b u l e s as e a r l y as  16 s i x t e e n hours f o l l o w i n g 4).  l i g a t i o n of the vasa e f f e r e n t i a  (table  T h i s i n c r e a s e was g r e a t e s t a f t e r t h e v a s a e f f e r e n t i a had  been l i g a t e d f o r seven d a y s . demonstrating  Only t h e m a j o r i t y o f t u b u l e s  a s i m i l a r degree o f a t r o p h y were c o n s i d e r e d .  TABLE  4  Mean Diameter o f 20 S e m i n i f e r o u s Tubules  From  Each P o s t -  o p e r a t i v e Stage F o l l o w i n g Vasa E f f e r e n t i a L i g a t i o n i n Mice Duration of ligation  Experimental d i a m e t e r (u)  Control diameter (u)  16 hour  205  1 day  195  172  2 day  210  184  4 day  205  176  5 day  215  179  7 day  220  187  The  ,  180  i n c r e a s e i n s i z e o f s e m i n i f e r o u s t u b u l e s was c o n -  current with a swelling  o f t h e l i g a t e d t e s t i s when compared  with the contralateral control.  The t u r g i d i t y was maximal  between 2 and 5 days p o s t l i g a t i o n .  By 7 days p o s t l i g a t i o n ,  the s i z e o f t h e l i g a t e d t e s t i s had decreased s i z e of the control  testis.  t o about t h e  17  C.  Changes i n t h e Number o f S e r t o l i C e l l N u c l e i Counts o f S e r t o l i c e l l n u c l e i f o r each of t h e c r o s s  s e c t i o n s of s e m i n i f e r o u s t u b u l e s used f o r t u b u l e measurements, showed a d e f i n i t e i n c r e a s e i n the number of n u c l e i a t 2 days postligation  (table 5).  This numerical increase i n n u c l e i  p e r s i s t e d u n t i l 5 days p o s t l i g a t i o n , when a marked d e c r e a s e in Sertoli c e l l nuclei resulted.  Counts o f S e r t o l i  cell  n u c l e i i n c o n t r a l a t e r a l t e s t e s showed no apparent change. T h i s n u m e r i c a l i n c r e a s e i n S e r t o l i c e l l n u c l e i was  a l s o observed  r a t s f o l l o w i n g l i g a t i o n of t h e v a s a e f f e r e n t i a ( p l a t e 9 ) . t h e s e a n i m a l s were i n j e c t e d s u b c u t a n e o u s l y  in  When  with colchicine,  however, no i n d i c a t i o n of S e r t o l i c e l l s u n d e r g o i n g m i t o s i s c o u l d be  detected. TABLE 5  Mean S e r t o l i C e l l Counts From 20 S e m i n i f e r o u s Tubule Cross S e c t i o n s F o l l o w i n g Vasa E f f e r e n t i a L i g a t i o n i n M i c e D u r a t i o n of l i g a t i o n 1  E x p e r i m e n t a l count  C o n t r o l count  day  8.7  8.5  2 day  13.0  8.9  4  day  16.8  7.8  5  day  13.4  8.4  7 day  7.6  9.3  18  D.  H i s t o l o g i c a l Changes i n t h e The f i r s t change observed  epididymis following decrease  Epididymis  i n c r o s s s e c t i o n s o f the  l i g a t i o n of t h e v a s a e f f e r e n t i a was  i n t h e number of sperm from t h a t observed  a  i n the  c o n t r a l a t e r a l c o n t r o l e p i d i d y m i s a t 2 days p o s t l i g a t i o n ( f i g . 115).  At 4 days p o s t l i g a t i o n  ( f i g . 116), some c r o s s s e c t i o n s  o f t h e d u c t midway between t h e caput e p i d i d y m i d i s and cauda e p i d i d y m i d i s were almost d e v o i d of spermatozoa, w h i l e o t h e r s c o n t a i n e d d i s o r g a n i z e d areas o f n e c r o t i c spermatozoa and PAS p o s i t i v e amorphous masses b e l i e v e d t o be mucus. o f a few d u c t s appeared n o r m a l .  The  contents  The c o n t e n t s of a l l d u c t  t i o n s appeared abnormal a f t e r 5 days p o s t l i g a t i o n  -  sec-  ( f i g . 117).  Many r e g i o n s o f t h e d u c t were d e v o i d o f spermatozoa. At 7 days postligation  ( f i g . 118), most r e g i o n s o f t h e d u c t midway a l o n g  t h e e p i d i d y m i s were d e v o i d o f spermatozoa or c o n t a i n e d s m a l l clumps o f n e c r o t i c spermatozoa and mucus.  E.  H i s t o l o g i c a l Changes i n the Germinal E p i t h e l i u m  The d e g e n e r a t i v e changes i n t h e g e r m i n a l e p i t h e l i u m were e x t r e m e l y v a r i e d i n d i f f e r e n t t u b u l e s ( f i g . 102).  I t was  19 not uncommon t o f i n d a m o r p h o l o g i c a l l y  normal, or s l i g h t l y  atrophic tubule adjacent t o a n e c r o t i c tubule  as l a t e as  4 days a f t e r t h e l i g a t i o n was made ( f i g . 1 0 3 ) . The degree of a t r o p h y i n each t u b u l e , however, g e n e r a l l y remained t h e same t h r o u g h o u t i t s l e n g t h .  This c o n d i t i o n  conveniently  lends i t s e l f t o a c l o s e a n a l y s i s of t h e process of atrophy at the d i f f e r e n t stages of the seminiferous generative  cycle.  The d e -  changes i n t h e g e r m i n a l e p i t h e l i u m a r e summed up  i n t a b l e 6, and a r e i l l u s t r a t e d on p l a t e s 3 - 6  ( f i g . 15 -  86). From t h e a n a l y s i s o f t h e d e g e n e r a t i v e changes a t each s t a g e o f t h e s e m i n i f e r o u s describe  a general  iferous tubule.  c y c l e , i t was p o s s i b l e t o  scheme f o r t h e d e g e n e r a t i o n o f a semin-  The scheme i s based on two d i f f e r e n t  h i s t o l o g i c a l p i c t u r e s found i n a t r o p h i c t u b u l e s : f i r s t , i n stages 1 - IX of t h e c y c l e  ( f i g . 2 - 10);  second, i n s t a g e s X - X l l o f t h e c y c l e  erature.  has not p r e v i o u s l y  the  ( f i g . 11 - 1 3 ) . A  diagram o f t h i s scheme i s shown i n f i g u r e 1 - A. proposed,  the  been d e s c r i b e d  The scheme  i n the  lit-  20  TABLE  6  The Histological Changes i n the Following Ligation of the Vasa  Postligation period  16 h o u r  Spermatogonia ( a l l stages)  normal  Spermatocyte s Resting (VI-Vlll)  normal  Secondary spermatocytes (stage X l l )  Pachytene ( a l l stages)  Leptotene & Zygotene (Vlll-Xll)  normal'  normal  normal  - n o r m a l i n most-, t u b u l e s - r e d u c e d nuBnberlin s t a g e s 1  day  normal  normal  1V-V1 (fig»"3&J40,46)  normal -a  2 day  4,days  5 days  normal  no changes detected  no changes detected  normal  no changes detected  no changes detected  normal  -many s t i l l normal -pyknotic c e l l s common -occasional sloughing  • l i t t l e change from 4 day postligation period  few " g h o s i ^ / ' j c e l l s (fig. 82):.  - r e d u c t i o n -:flii .[ number (fig. 23^^,41,59) - s l o u g h i n g du&liig s t a g e s 1,1V,V11,^:X11 -increase iii':*' ghost" c e l l s (flg^2^i53)  no changes detected  • l i t t l e change from 5 day postligation period  ". V '  -normal i n most t u b u l e s -increased sloughing ( f i g .  -most h a v e b e e n released prematurely i n t o t h e lumen ( f i g . 16,70,76)  58)  - r e d u c t i o n i n number -occurrence of chromatin-ringed n u c l e i ( f i g . 35,53) -increased sloughing ( f i g . 35,65 m - f o r m a t i o n o f MNB ( f i g . 1 7 , 5 9 )  -greater reduction i n number - o n l y a few divisions (fig. 85)  - g r e a t e r r e d u c t i o n i n number -sloughing ( f i g . 31,37,55,61) - c h r o m a t i n - r i n g e d n u c l e i common (fig. 55) - r e d u c t i o n i n number o f MNB ( f i g . 55,61)  • l i t t l e change from 5 day postligation period  - g r e a t e r r e d u c t i o n i n number - s l o u g h i n g i n most t u b u l e s (fig. 20,26,32,56,62)  ;  (fig. 73%ij*0  73)  &g£$gij>  >.*•'<•  -  '  ( f i g . 20g .-...-; -little changefrom 5 day posti^^itiLon p e r i o d %  ;  -  111'  -greater reduction in number (fig•j 31,61) - a b s e n t inl/S^e t u b u l e s (fig. 4 9 J J ' ^ ; i . - "ghost" ^'JULs* I common -nuclear fca^meiitation  18',$'$$)  I'V"'-  Spermatids i n steps 10 - 16 of spermiogenesis (stages X — X l l & 1 - Vlll) -sloughing at IV - V l l l  - i n c r e a s e ih.nuipber of "ghost" -of ten in;congested lumina ( f i g . i 72)  (fig,  'it  -normal i n most t u b u l e s -occasional sloughing  - g r e a t e r r e d u c t i o n i n number -increase i n sloughing ( f i g . 36,48,54,60) - i n c r e a s e d number o f c h r o m a t i n ringed n u c l e i ( f i g . 42,54,66)  i n number  - a b s e n t in&s&met' t u b u l e s 7 days  reduction in number (fig. 83)  . v-ra. •  Spermatids i n steps 1 - 9 of spermiogenesis ( s t a g e s 1 - IX)  -greater reduction i n number -sloughing -numerous " g h o s t " cells (fig. 84)  -reduction  -s loughing  no changes detected  normal (fig. 82)  Germinal Epithelium Efferentia in Mice  '•off:  0 r \: - •  IV  stages  - s l o u g h i n g i n most tubules (fig. 17, 23,47,71,77,83)  - v e r y few r e m a i n (fig. 24,30,36,42, 4 8 , 54, 78) -completely b l o c k the l u m i n a o f some tubules (fig. 72) -absent or g r e a t l y r e d u c e d i n number i n most t u b u l e s (fig. 25,31,55,61 79,85) -sloughing i n tubules r e t a i n i n g these c e l l s - l i t t l e change from t h a t observed at 5 days p o s t l i g a t i o n  Sertoli cells ( a l l stages)  normal  normal  - i n c r e a s e i n number o f i n some t u b u l e s  nuclei  -numerical increase i n nuclei(fig.78 - n u c l e a r m i g r a t i o n s t o w a r d lumen (fig. 24,72,78) -some n u c l e i i n l u m i n a ( f i g . 72) - n u c l e a r shape conforms t o edges o f v a c u o l e s , MNB ( f i g . 7 2 , 7 8 )  -numerous n u c l e i a r e sloughed into lumina-(fig. 53,73), otherwise, similar to that observed at 4 days p o s t l i g a t i o n  -number o f n u c l e i r e d u c e d t o about t h a t of c o n t r o l t e s t i s -most n u c l e i l o c a t e d a t basement membrane  21  D e g e n e r a t i v e Steps o f a S e m i n i f e r o u s Tubule  Stages 1 - IX o f t h e C y c l e 1. Spermatid Sloughing Stage  2. M u l t i n u c l e a t e d Body Stage  3. S e r t o l i C e l l Cytoplasm Stage 1.  Cellular Sloughing Stage  4. S e r t o l i C e l l > Sloughing Stage  2• Spermatocyte Ghost C e l l Stage  Stages X - X l l o f t h e C y c l e  Fig.  1 - A The two v a r i a t i o n s o f d e g e n e r a t i o n i n s e m i n i f e r o u s  t u b u l e s a r e caused by t h e presence o f d i f f e r e n t  t y p e s o f germ  c e l l s i n the r e s p e c t i v e stages of the c y c l e of the seminiferous epithelium.  D u r i n g t h e i n i t i a l phase o f t e s t i c u l a r  atrophy i n  s t a g e s 1 - IX o f t h e c y c l e , t h e m a j o r i t y o f c e l l s i n t h e germi n a l e p i t h e l i u m a r e s p e r m a t i d s i n s t e p s 1 t o 9 of s p e r m i o g e n e s i s ; i n s t a g e s X - X l l o f t h e c y c l e , t h e m a j o r i t y of c e l l s a r e spermatocytes•  22  1.  Degenerative The  Steps i n Stages X - X l l o f t h e C y c l e  i n i t i a l step i n the degeneration  of seminiferous  t u b u l e s i n s t a g e s X - X l l o f t h e c y c l e has been termed t h e " c e l l u l a r s l o u g h i n g " stage  ( f i g . 88).  During t h i s p e r i o d of  c e l l u l a r e x f o l i a t i o n , most o f t h e s p e r m a t i d s  and some s p e r -  m a t o c y t e s a r e r e l e a s e d i n t o t h e lumen, l e a v i n g m a i n l y  sper-  matocytes i n t h e g e r m i n a l e p i t h e l i u m t o undergo f u r t h e r degeneration. S e m i n i f e r o u s t u b u l e s i n t h e n e x t stage i n t u b u l a r d e g e n e r a t i o n , t h e "spermatocyte g h o s t c e l l " stage  ( f i g . 89,  9 0 ) , a r e c h a r a c t e r i z e d b y pachytene and d i p l o t e n e spermatoc y t e s w i t h p y k n o t i c n u c l e i and a c y t o p l a s m i c a f f i n i t y f o r p e r i o d i c acid - S c h i f f reagent. a r e sloughed  Some o f t h e s e  spermatocytes  i n t o t h e lumen ( f i g . 8 9 ) , b u t most undergo d e -  generation i n the germinal epithelium.  Severely n e c r o t i c  s p e r m a t o c y t e s have been termed "ghost c e l l s " as t h e y have l i t t l e o r no n u c l e a r m a t e r i a l p r e s e n t , and have a reduced c y t o p l a s m i c a f f i n i t y f o r PAS ( f i g . 9 0 ) .  V e r y few s m a l l m u l t i -  n u c l e a t e d b o d i e s a r e formed by spermatocytes.  The younger  g e n e r a t i o n o f l e p t o t e n e and zygotene spermatocytes appear m o r p h o l o g i c a l l y r e s i s t a n t a t t h e b e g i n n i n g o f t h e "ghost c e l l " s t a g e , b u t many undergo p y k n o s i s d u r i n g t h e l a t e phases o f  23 t h i s degenerative stage.  Secondary spermatocytes  i n stage  X l l o f t h e c y c l e a r e e i t h e r sloughed i n t o t h e lumen or undergo d i v i s i o n .  Many d i v i d i n g spermatocytes  are i n v a r i o u s  s t a t e s of k a r y o r r h e x i s and have a c q u i r e d a PAS cytoplasm.  - positive  Numerous v a c u o l e s are f o r m e d \ i n t h e g e r m i n a l  epithelium.  The number of S e r t o l i c e l l n u c l e i i n c r e a s e d u r i n g  t h i s s t a g e , and some " m i g r a t e " away from t h e basement membrane. The n e x t two d e g e n e r a t i v e s t a g e s , t h e " S e r t o l i  cell  c y t o p l a s m " s t a g e , and t h e " S e r t o l i c e l l s l o u g h i n g " s t a g e , are analogous t o t h e l a s t two d e g e n e r a t i v e s t a g e s t h a t occur i n t u b u l a r a t r o p h y d u r i n g s t a g e s 1 - IX o f t h e c y c l e ( f i g . 98,99).  2.  D e g e n e r a t i v e Steps i n Stages 1 - IX of t h e C y c l e The d e g e n e r a t i v e changes o c c u r r i n g i n s t a g e s 1 - IX  of  t h e c y c l e g e n e r a l l y f o l l o w t h e same sequence of e v e n t s .  The i n i t i a l s t e p i s t h e " s p e r m a t i d s l o u g h i n g " s t a g e , c h a r a c t e r i z e d by a g e n e r a l l o o s e n i n g o f t h e g e r m i n a l e p i t h e l i u m , and r e s u l t i n g i n s l o u g h i n g of most of t h e o l d e r g e n e r a t i o n o f s p e r m a t i d s i n s t e p s 13 t o 16 of s p e r m i o g e n e s i s Spermatids i n s t e p s 15 and 16  ( f i g . 92).  (stages V - V l l l of the c y c l e )  a r e g e n e r a l l y t h e f i r s t t o be sloughed i n t o t h e lumen because of t h e i r c l o s e p r o x i m i t y t o t h e lumen.  Some o f t h e younger  24 generation of spermatids i n steps 1 t o 9 of spermiogenesis a r e a l s o sloughed i n t o t h e lumen ( f i g . 9 1 ) . D u r i n g t h e l a t t e r p a r t o f t h e " s p e r m a t i d s l o u g h i n g " s t a g e , some o f t h e n u c l e i of  t h e younger g e n e r a t i o n o f s p e r m a t i d s undergo p y k n o s i s ,  producing nuclear r i n g formations of chromatin. of 92),  Some r e g i o n s  seminiferous t u b u l e s c o n t a i n a l l n e c r o t i c spermatids ( f i g . b u t g e n e r a l l y , t h e p r o p o r t i o n o f normal t o n e c r o t i c s p e r -  matids i s v a r i a b l e .  There does n o t seem t o be any c o r r e l a t i o n  between t h e number o f n e c r o t i c s p e r m a t i d s and t h e s t a g e o f t h e cycle of the seminiferous epithelium.  Some p r i m a r y  spermato-  c y t e s i n pachytene undergo p y k n o s i s and a c q u i r e a c y t o p l a s m i c a f f i n i t y f o r PAS.  S e r t o l i c e l l n u c l e i i n c r e a s e i n number and  some a r e d i s p l a c e d away from t h e basement membrane. The f u s i o n o f n u c l e a r membranes,' e i t h e r a t t h e a c r o somic o r o t h e r r e g i o n s o f a d j a c e n t s p e r m a t i d n u c l e i , i n normal or n e c r o t i c s p e r m a t i d s , i n i t i a t e s t h e n e x t s t a g e o f a t r o p h y , t h e " m u l t i n u c l e a t e d body" s t a g e ( f i g . 93, 9 4 ) .  Initially,  many s m a l l m u l t i n u c l e a t e d b o d i e s (MNB) c o n t a i n i n g o n l y s e v e r a l s p e r m a t i d n u c l e i a r e formed, b u t t h e s e s m a l l e r MNB t e n d t o f u s e t o g e t h e r t o form r e l a t i v e l y fewer b u t much l a r g e r MNB ( f i g . 9 5 ) . Pachytene  and some l e p t o t e n e spermatocytes undergo d e g e n e r a t i v e  changes as d e s c r i b e d i n t h e "spermatocyte g h o s t c e l l " s t a g e o f t u b u l e s i n s t a g e s X - X l l o f t h e c y c l e ( f i g . 8 9 ) . MNB c o n t a i n ing  spermatocytes  a r e v e r y seldom formed.  As t h e s e MNB and  25  n e c r o t i c spermatocytes d i s a p p e a r , v a c u o l e s o f v a r y i n g s i z e s are l e f t i n the germinal e p i t h e l i u m ( f i g . 97). O c c a s i o n a l l y , t h e g e r m i n a l e p i t h e l i u m o f some t u b u l e s i s g r e a t l y  reduced  i n c e l l u l a r c o n t e n t , and t h e lumen e x t r e m e l y c o n j e s t e d w i t h spermatozoa,  l o o s e c e l l s and c e l l u l a r d e b r i s ( f i g . 9 6 ) •  D u r i n g t h e MNB  s t a g e , S e r t o l i c e l l s appear a c t i v e l y  i n phagocytosis:  numerous " m i g r a t i n g " S e r t o l i c e l l  a r e l o c a t e d near o r a t t h e edges o f v a c u o l e s , MNB, spermatocytes.  engaged nuclei and a t r o p h i c  The c y t o p l a s m o f t h e S e r t o l i c e l l s toward t h e  end of t h i s s t a g e becomes r e l a t i v e l y f r e e o f c e l l u l a r components ( f i g . 97). The s u c c e e d i n g " S e r t o l i c e l l c y t o p l a s m " s t a g e o f a t r o p h y ( f i g . 98) i s a marked change from t h e p r e v i o u s MNB stage.  The g e r m i n a l e p i t h e l i u m c o n t a i n s e s s e n t i a l l y  spermatogonia resting,  a d j a c e n t t o t h e basement membrane; o c c a s i o n a l  l e p t o t e n e o r e a r l y pachytene p r i m a r y spermatocytes  some o f which appear m o r p h o l o g i c a l l y n o r m a l ; matids;  only  phagocytized c e l l u l a r debris;  number o f S e r t o l i ; c e l l n u c l e i , from t h e basement membrane.  -  v e r y few s p e r -  and an i n c r e a s e d  many of which a r e l o c a t e d away  Many lumina o f s e m i n i f e r o u s t u b -  u l e s a t t h i s s t a g e o f a t r o p h y a r e o c c u p i e d by S e r t o l i  cell  c y t o p l a s m , so t h a t f r e q u e n t l y t h e lumina a r e almost n o n e x i s t ent ( f i g . 9 7 ) .  26  As a t r o p h i c c o n d i t i o n s become more s e v e r e , germinal  e p i t h e l i u m becomes c o r r e s p o n d i n g l y  the  t h i n n e r as a  r e s u l t of " S e r t o l i c e l l s l o u g h i n g " - t h e n e x t s t a g e i n t h e sequence o f a t r o p h i c s t e p s  ( f i g . 99).  During t h i s p e r i o d ,  l a r g e masses of v a c u o l a t e d  S e r t o l i c e l l s , phagocytozed i n -  c l u s i o n s , and o c c a s i o n a l l y , s p e r m a t i d s and s p e r m a t o c y t e s are sloughed i n t o t h e r e l a t i v e l y empty t u b u l a r lumen.  This  ex-  f o l i a t i o n appears t o be caused by e x t e n s i v e v a c u o l a t i o n i n t h e S e r t o l i c e l l c y t o p l a s m near t h e basement membrane ( f i g . 100), w h i c h l e a d s e v e n t u a l l y t o a s e p a r a t i o n from t h e r e m a i n der of t h e g e r m i n a l e p i t h e l i u m a d j a c e n t t o t h e basement membrane.  E x f o l i a t e d S e r t o l i c e l l s i n t h e lumens of s e m i n i f e r -  ous t u b u l e s  ( f i g . 101)  undergo c e l l u l a r d e g e n e r a t i o n  o t h e r c e l l s which become s e p a r a t e d elium.  as  do  from t h e g e r m i n a l e p i t h -  As a r e s u l t of the s l o u g h i n g , t h e g e r m i n a l  epithelium  becomes g r e a t l y reduced i n s i z e and c o n s i s t s o n l y o f spermatog o n i a , S e r t o l i c e l l s , and the o c c a s i o n a l p r i m a r y s p e r m a t o c y t e .  3.  The  F a t e of M u l t i n u c l e a t e d B o d i e s  The most s t r i k i n g d e v i a t i o n from t h e normal t u b u l a r morphology d u r i n g a t r o p h y i s the d i s p o s i t i o n of s p e r m a t i d s i n stages  1 - IX of t h e c y c l e i n b o t h r a t s and mice by  formation  27  of m u l t i n u c l e a t e d b o d i e s  (MNB).  Most of t h e s e MNB  degeneration i n the germinal e p i t h e l i u m .  undergo  Four s t a g e s of  MNB  d e g e n e r a t i o n can be d i f f e r e n t i a t e d w i t h i r o n h a e m a t o x y l i n . I n i t i a l l y , MNB spermatid n u c l e i  c o n t a i n m o r p h o l o g i c a l l y normal  ( f i g . 94), n e c r o t i c spermatid n u c l e i , or a  c o m b i n a t i o n of b o t h .  The i n f r e q u e n t o c c u r r e n c e of t h e s e  MNB  c o n t a i n i n g a p p a r e n t l y normal s p e r m a t i d s suggests t h a t normal n u c l e i degenerate MNB  r a p i d l y i n MNB  to nuclear r i n g formations.  t y p i c a l l y c o n s i s t of s p e r m a t i d n u c l e i w i t h i n t a c t mem-  branes  l o c a t e d a t t h e p e r i p h e r y , and a c e n t r a l c y t o p l a s m i c  mass ( f i g . 104, 105).  Stage 1 MNB  c o n t a i n t h e most h y p e r -  chromatic spermatid n u c l e i . A marked r e d u c t i o n i n n u c l e a r and c y t o p l a s m i c s t a i n i n t e n s i t y c h a r a c t e r i z e s s t a g e 2 MNB  ( f i g . 104,  105).  The n u c l e a r membranes of t h e s p e r m a t i d s a r e s w o l l e n b u t intact• Stage 3 MNB  ( f i g . 104) a r e c h a r a c t e r i z e d by a  d i s r u p t i o n of t h e s p e r m a t i d n u c l e a r membranes c a u s i n g a r e d u c t i o n of n u c l e a r s t a i n i n g .  Stage 3 MNB  "multinucleated ghosts"  L a t e s t a g e s o f MNG  (MNG).  greatly disorganized ( f i g .  have been termed become  105).  The f i n a l s t a g e o f MNB  n e c r o s i s i n v o l v e s complete  l y s i s of t h e r e m a i n i n g c e l l u l a r mass.  The d i s a p p e a r a n c e  of  28 t h e MNG  from t h e g e r m i n a l e p i t h e l i u m i s c o n c u r r e n t w i t h t h e  formation of vacuoles w i t h i n the germinal e p i t h e l i u m ( f i g . O c c a s i o n a l l y , MNB  are sloughed i n t o t h e t u b u l e lumen d u r i n g  any one of t h e 4 s t a g e s o f d e g e n e r a t i o n  4.  105).  (fig.  105).  The F a t e of R e s i d u l e B o d i e s Residual cytoplasmic bodies  (fig.  by s p e r m a t i d s a t s t e p 16 of s p e r m i o g e n e s i s  106), d i s c a r d e d  i n t h e mouse, and  s t e p 19 i n t h e r a t ( s t a g e s V l l and V l l l o f t h e c y c l e ) , a s i m i l a r d e g e n e r a t i v e p r o c e s s as MNB germinal epithelium.  follow  d u r i n g atrophy of the  I n o r d e r t o observe t h e d e g e n e r a t i o n of  r e s i d u a l b o d i e s , s e m i n i f e r o u s t u b u l e s were l o c a t e d i n w h i c h s p e r m i o g e n e s i s had c o n t i n u e d t o s t e p 16  ( s t e p 19 i n t h e r a t )  b u t a t w h i c h p o i n t , a t r o p h i c c o n d i t i o n s of t h e p a r t i c u l a r t u b u l e had i n h i b i t e d f u r t h e r s p e r m a t i d development. t u b u l e s i n i t i a l l y c o n t a i n MNB  comprised  These  of s t a g e V l l or  s p e r m a t i d s , and l a r g e numbers o f r e s i d u a l b o d i e s  (RB)  randomly i n t h e c y t o p l a s m o f S e r t o l i c e l l s  107).  (fig.  Vlll  distributed The  RB  a r e t h e n o r g a n i z e d i n t o " r e s i d u a l body masses" (RBM)  so t h a t  o n l y o c c a s i o n a l l y a r e RB seen a l o n e i n S e r t o l i c e l l s  (fig.  109),  RBM  108,  undergo a s i m i l a r l y s i s as m u l t i n u c l e a t e d b o d i e s ,  l e a v i n g r e s i d u a l vacuoles i n the germinal e p i t h e l i u m .  29  F.  A t r o p h i c R e a c t i o n s i n Normal T i s s u e In n e a r l y a l l c o n t r o l t e s t e s a n a l y z e d , t h e h i s t o -  l o g i c a l appearance o f the g e r m i n a l e p i t h e l i u m appeared n o r m a l . An e x c e p t i o n o c c u r r e d i n a c o n t r o l mouse t e s t i s t a k e n a t 1 day p o s t l i g a t i o n . . The t e s t i c u l a r h i s t o l o g y appeared normal i n one p a r t i c u l a r s e m i n i f e r o u s t u b u l e a t c o n s e c u t i v e X l l and 1 o f t h e c y c l e .  The r e m a i n i n g  of t h e t u b u l e appeared n o r m a l .  except  stages  s t a g e s a l o n g the l e n g t h  T u b u l a r c r o s s s e c t i o n s of t h e  a t r o p h i c r e g i o n c o n t a i n i n g these 2 stages r e v e a l e d m u l t i n u c l e a t e d b o d i e s c o n s i s t i n g o f secondary i n t e r p h a s e spermatoc y t e s at stage X l l of the c y c l e ( f i g . I l l , spermatids  a t s t a g e 1 ( f i g . 110).  These MNB  112), and s t e p 1 occupied only a  p a r t i a l r e g i o n of t h e t u b u l e , w h i l e t h e remainder of t h e t u b u l e appeared n o r m a l .  Stage 1 s p e r m a t i d s  and secondary spermato-  c y t e s i n i n t e r p h a s e were a l s o seen i n t h e lumen.  The  other  germ c e l l s of t h e g e r m i n a l e p i t h e l i u m appeared u n a f f e c t e d .  30  DISCUSSION  The r e s u l t s of t h e p r e s e n t s t u d y on s e m i n i f e r o u s t u b u l e d e g e n e r a t i o n c l e a r l y show t h e f o l l o w i n g :  (1)  the  t i m i n g o f t h e c y c l e does n o t appear t o be d i s r u p t e d : a t a l l p o s t o p e r a t i v e p e r i o d s , t h e v i a b l e germ c e l l s are a s s o c i a t e d w i t h t h e same s t a g e s of t h e c y c l e as t h e y a r e i n t h e testis,  (2)  normal  t h e f r e q u e n c y w i t h which each s t a g e of t h e c y c l e  o c c u r s i n t h e mouse (not measured i n t h e r a t ) does n o t change, (3)  a d e f i n i t e sequence of n e c r o t i c e v e n t s p e c u l i a r t o each  s t a g e o f t h e c y c l e o c c u r s , and  (4)  t h e number and p o s i t i o n  of t h e S e r t o l i c e l l n u c l e i change. One of t h e main p r o p e r t i e s o f t h e normal t e s t i s i s t h e s t a b i l i t y of t h e s e m i n i f e r o u s c y c l e .  The mechanism f o r  t h i s e x a c t t i m i n g d u r i n g t h e p r o l i f e r a t i o n of germ c e l l s n o t y e t been demonstrated.  P r e v i o u s s t u d i e s , however, on  t e s t i c u l a r a t r o p h y i n d u c e d by l e s s s e v e r e methods and M o r g e n t a l e r , 1955)  has  i n hypophysectomized  f o l l o w i n g an i n c r e a s e i n t h e i n t r a t e s t i c u l a r  (Clermont  r a t s , and i n r a t s temperature  (Chowdhury and S t e i n b e r g e r , 1964), agree w i t h t h e p r e s e n t o b s e r v a t i o n s i n mice ( t a b l e 3) t h a t t h e f r e q u e n c y d i s t r i b u t i o n o f t h e s t a g e s o f t h e c y c l e does n o t change.  Under t h e  31 e x t e n s i v e d e g e n e r a t i v e c o n d i t i o n s of t h e p r e s e n t  experiments,  t h e n , t h e p e r s i s t e n c e o f t h e same c e l l u l a r a s s o c i a t i o n s ,  and  t h e same f r e q u e n c y d i s t r i b u t i o n o f s t a g e s suggests t h a t  the  r e g u l a t i o n of t h e c y c l e i s an i n h e r e n t g e n e t i c c o n t r o l .  Such  a c o n t r o l would account f o r r e g i o n s i n a t r o p h i c t u b u l e s which c o n t a i n n e c r o t i c s p e r m a t i d s and spermatocytes  beside apparently  normal c e l l u l a r c o u n t e r p a r t s . L i g a t i o n of t h e v a s a e f f e r e n t i a must a f f e c t t h e g e r m i n a l e p i t h e l i u m i n a n o n s p e c i f i c manner s i n c e d e g e n e r a t i v e changes t o s p e r m a t i d s and spermatocytes  occurred i n v a r i a b l e  numbers a t a l l s t a g e s of t h e c y c l e ( t a b l e 6 ) • t h i s a t r o p h y has been suggested by Smith  The cause of  (1962) t o be t h e r e -  s u l t o f i n c r e a s e d i n t r a t e s t i c u l a r p r e s s u r e due t o t h e accumul a t i o n o f i n t r a t u b u l a r f l u i d and g e r m i n a l p r o d u c t s .  T h i s would  account f o r t h e i n c r e a s e i n t h e s i z e of t h e s e m i n i f e r o u s t u b u l e s s h o r t l y a f t e r t h e l i g a t i o n was made ( t a b l e 4 ) . of  A total  blockage  t h e e x c u r r e n t d u c t s was v e r i f i e d f o r each t e s t i s by o b s e r v i n g  a decrease i n the contents of the epididymal duct  ( f i g . 113  -  118). P r e v i o u s s t u d i e s on n o n s p e c i f i c d e g e n e r a t i o n o f t h e g e r m i n a l e p i t h e l i u m i n d u c e d by o t h e r methods, have r e p o r t e d a t r o p h i c c o n d i t i o n s of the t e s t i s at v a r i o u s time f o l l o w i n g t h e i n i t i a l i n d u c t i o n of t h e a t r o p h y  intervals  (artificial  32 cryptorchidism:  C l e g g , 1963 a,b;  Niemi and Korraano, 1965; tures:  D a v i s and F i r l i t ,  1966;  exposure of t e s t i s t o h i g h tempera-  S t e i n b e r g e r and D i x o n , 1959;  O e t t l e and H a r r i s o n , 1952).  t e s t i c u l a r ischaemia:  The d i f f i c u l t y i n i n t e r p r e t i n g  t h e s e r e s u l t s i s t h a t t h e r a t e a t Which d i f f e r e n t s e m i n i f e r o u s t u b u l e s undergo d e g e n e r a t i o n v a r i e s , c r e a t i n g numerous s t a t e s of a t r o p h y .  The p r e s e n t d e s c r i p t i o n o f t h e d e g e n e r a t i v e s t e p s  which a s e m i n i f e r o u s t u b u l e undergoes a t each s t a g e o f t h e s e m i n i f e r o u s c y c l e i s t h e r e f o r e an i m p o r t a n t g u i d e .  By u s i n g  t h i s i n f o r m a t i o n , t h e c o u r s e of p r e v i o u s d e g e n e r a t i v e changes can be d e t e r m i n e d i n any n e c r o t i c t u b u l e . The c l a s s i f i c a t i o n of t h e s t e p s o f s e m i n i f e r o u s t u b u l e d e g e n e r a t i o n i n t h e mouse i s based on t h e t y p e o f a t r o p h i c r e a c t i o n which a f f e c t s t h e m a j o r i t y o f germ c e l l s a t each s t a g e of t h e c y c l e ( f i g . 1 - A ) .  A c c o r d i n g l y , t h e s t a g e s of  t h e c y c l e were d i v i d e d i n t o two main groups: Group 1, c o n s i s t i n g of s t a g e s 1 - IX of t h e c y c l e ; and Group 2, c o n s i s t i n g of s t a g e s X - X l l (X - XIV i n t h e r a t ) .  The d i v i s i o n i n t o t h e  2 groups o f s t a g e s i s a r e s u l t o f t h e m o r p h o l o g i c a l changes i n t h e s p e r m a t i d n u c l e u s d u r i n g l a t e s t a g e IX o f t h e c y c l e .  The  marked t r a n s f o r m a t i o n of t h e r e l a t i v e l y round s p e r m a t i d n u c l e u s t o t h e e l o n g a t e c u r v e d head of t h e spermatozoon appears  to  33 a l t e r t h e response o f t h e more mature s p e r m a t i d s .  Instead of  f o r m i n g m u l t i n u c l e a t e d b o d i e s as t h e younger g e n e r a t i o n of s p e r m a t i d s do d u r i n g a t r o p h i c c o n d i t i o n s , t h e o l d e r s p e r m a t i d s a r e sloughed i n t o t h e lumen - l e a v i n g t h e s p e r m a t i d s i n s t a g e s 1 - I X , and t h e spermatocytes n e c r o t i c changes.  i n s t a g e s X - X l l t o undergo  Seminiferous t u b u l e s s i m i l a r t o those des-  c r i b e d i n t h e " S e r t o l i c e l l c y t o p l a s m " s t a g e ( f i g . 9 8 ) , have been r e p o r t e d i n r a t t e s t i s f o l l o w i n g 30 days of a r t i f i c i a l l y i n d u c e d c r y p t o r c h i d i s m ( C l e g g , 1963;  D a v i s and F i r l i t ,  1966).  Tubules o f t h e " S e r t o l i c e l l s l o u g h i n g " s t a g e ( f i g . 99,  100),  however, have n o t been r e p o r t e d b e f o r e i n t e s t e s s u b j e c t e d t o e i t h e r l i g a t i o n o f t h e v a s a e f f e r e n t i a or a r t i f i c i a l l y  induced  cryptorchidism. In t u b u l e s observed beyond t h e " m u l t i n u c l e a t e d body" s t a g e ( f i g . 104) or t h e "spermatocyte ghost c e l l " stage ( f i g . 89, 90) of t u b u l e d e g e n e r a t i o n , pachytene p r i m a r y  spermatocytes,  i n g r e a t l y reduced numbers, were t h e most advanced s t a g e s of germ c e l l s i n t h e g e r m i n a l e p i t h e l i u m . T h i s has a l s o been noted i n c r y p t o r c h i d r a t t e s t e s w h i c h have undergone s e v e r e d e g e n e r a t i o n (Davis and F i r l i t ,  1966).  The p r o c e s s of spermatogenesis  would  t h e r e f o r e appear t o be i n h i b i t e d from p r o c e e d i n g beyond t h e pachytene  s t a g e o f p r i m a r y spermatocytes i n t u b u l e s which  are  34 w e l l i n t o or p a s t t h e above 2 s t e p s o f d e g e n e r a t i o n . In a l l s t e p s o f t u b u l a r  atrophy, the degeneration of  germ c e l l s i s i n t i m a t e l y a s s o c i a t e d of t h e S e r t o l i c e l l s .  w i t h an i n c r e a s e d  A consideration  e r a t i o n on S e r t o l i c e l l s i s t h e r e f o r e  activity  o f t h e e f f e c t s o f degenimportant.  The p r e s e n t  e x p e r i m e n t s i n d i c a t e t h a t 3 marked changes o c c u r t o S e r t o l i c e l l s during atrophy:  a numerical increase  i n their nuclei  ( t a b l e 5 ) , a m i g r a t i o n o f t h e i r n u c l e i away from t h e basement membrane ( f i g . 9 8 ) , and f i n a l l y ,  a d e c r e a s e i n t h e number o f  their nuclei. In normal t e s t e s o f r a t s and m i c e , t h e complement o f S e r t o l i c e l l s i s r e a c h e d s h o r t l y a f t e r b i r t h and m i t o t i c d i v i s i o n s c e a s e ( S a p s f o r d , 1962).  A few i n v e s t i g a t i o n s on t e s t i -  c u l a r a t r o p h y though, have r e p o r t e d an i n c r e a s e  i n t h e number  of S e r t o l i c e l l n u c l e i i n d e g e n e r a t i n g s e m i n i f e r o u s t u b u l e s ( i n hypophysectomized r a t s :  Clermont and M o r g e n t a l e r , 1955;  i n hypophysectomized r a t s t r e a t e d w i t h f o l l i c l e hormone: rat  Murphy, 1965;  testes:  i n a r t i f i c i a l l y induced  C l e g g , 1963).  stimulating cryptorchid  Clermont and M o r g e n t a l e r o f f e r no  explanation f o r t h e i r observations, but Clegg a t t r i b u t e s the numerical increase  of S e r t o l i c e l l n u c l e i t o a m i t o t i c d i v i s i o n s .  Recent e x p e r i m e n t s w i t h r a t c r y p t o r c h i d Firlit  t e s t e s by D a v i s and  (1966), however, i n d i c a t e o n l y an i n c r e a s e  i n Sertoli  35 c e l l size.  There have a l s o been p r e v i o u s r e p o r t s ,  according  t o N e b e l and Murphy (1960), o f an apparent m i t o t i c p r o l i f e r a t i o n of S e r t o l i c e l l s f o l l o w i n g i r r a d i a t i o n N e b e l and Murphy a t t r i b u t e t h i s i n c r e a s e the f o r m a t i o n of " r e s t i t u t i o n " t o i r r a d i a t e d t e s t e s , and  cells.  f o r e s t i l l i n doubt.  The  in Sertoli cells  These c e l l s are  The  arrest  possibility  during t e s t i c u l a r  to  peculiar  are produced as a r e s u l t of an  a t metaphase of. p r i m a r y s p e r m a t o c y t e s . Sertoli cell proliferation  of r a t t e s t e s .  of  atrophy i s there-  mechanism f o r t h e n u m e r i c a l  increase  i n n u c l e i observed i n t h e p r e s e n t e x p e r i m e n t s has not y e t been determined.  The  p o s s i b i l i t y of m i t o t i c p r o l i f e r a t i o n  S e r t o l i c e l l s however, cannot be r u l e d out by the  of  apparent  absence of m i t o t i c f i g u r e s i n S e r t o l i c e l l n u c l e i i n r a t treated with colchicine. t h y m i d i n e are now The  being carried  possible stimulus  c e l l behaviour during increase  Further investigations using  testes  tritiated  out. f o r the changes i n the  atrophic conditions  c o u l d be the  Sertoli large  i n p h a g o c y t i c m a t e r i a l i n g e s t e d by S e r t o l i c e l l s .  The  p h a g o c y t i c c a p a c i t y of S e r t o l i c e l l s i s w e l l known i n normal testes  (Smith and  atrophic testes  Lacy, 1959; (Lacy, 1960,  N i e m i and Kormano, 1965) 1962;  In S e r t o l i c e l l s undergoing a c t i v e  Reddy and  and  Svoboda, 1967).  p h a g o c y t o s i s of d e g e n e r a t i n g  36 germ c e l l s , Reddy and Svoboda have demonstrated  an immediate  i n c r e a s e i n t h e number o f lysosomes, and a 40 p e r c e n t i n c r e a s e i n a c i d phosphatase  content.  The r o l e o f S e r t o l i c e l l s d u r i n g  t e s t i c u l a r a t r o p h y t h e n , seems t o be d i r e c t e d m a i n l y toward p h a g o c y t o s i s o f d e g e n e r a t i n g germ c e l l s . One o b s e r v a t i o n o f S e r t o l i c e l l b e h a v i o u r i n t h e p r e s e n t e x p e r i m e n t s which does i n d i c a t e a change i n t h e i r r e a c t i o n t o i n g e s t e d m a t e r i a l , i s t h e i r d i s p o s i t i o n of r e s i d u a l bodies.  D u r i n g normal s p e r m a t o g e n e s i s , most r e s i d u a l b o d i e s  a r e r e l e a s e d i n t o t h e lumen.  A few a r e r e t a i n e d i n t h e germ-  i n a l e p i t h e l i u m , p h a g o c y t i z e d by S e r t o l i c e l l s and m i g r a t e toward t h e basement membrane (Niemi and Kormano, 1965; 1959;  D i e t e r t , 1966).  Some m i g r a t e i n d e p e n d e n t l y t o t h e  p e r i p h e r y o f t h e t u b u l e v i a i n t e r c e l l u l a r spaces D a v i s , 1965).  Lacy,  ( F i r l i t and  During seminiferous tubule degeneration, r e s i d -  u a l b o d i e s a r e p h a g o c y t i z e d i n g r e a t e r numbers by S e r t o l i c e l l s , b u t f a i l t o m i g r a t e t o t h e basement membrane.  Instead,  t h e y a r e o r g a n i z e d i n t o " r e s i d u a l body masses" ( f i g . 107, 108, 109). The i n c r e a s e i n S e r t o l i n u c l e i c o i n c i d e s w i t h t h e n u c l e a r " m i g r a t i o n s " o f S e r t o l i c e l l s away from t h e basement membrane.  Whether S e r t o l i c e l l s become s e p a r a t e d from t h e  37  basement membrane d u r i n g t h e i r n u c l e a r " m i g r a t i o n s " has n o t been determined.  A p o s s i b i l i t y - i f the i n c r e a s e i n S e r t o l i  n u c l e i i s by m i t o s i s , i s t h a t t h e newly formed S e r t o l i  cells  are d i s p l a c e d toward t h e lumen and do n o t come i n t o c o n t a c t w i t h t h e basement membrane.  During severe a t r o p h i c c o n d i t i o n s  then, these " m i g r a t i n g " S e r t o l i c e l l s would be sloughed  into  the lumen ( f i g . 99, 100). S e r t o l i c e l l s l o u g h i n g would a c count f o r t h e n u m e r i c a l decrease i n S e r t o l i c e l l n u c l e i a t 7 days p o s t l i g a t i o n  ( t a b l e 5 ) , and c o u l d a l s o account f o r  Clegg*s o b s e r v a t i o n of a r e d u c t i o n i n S e r t o l i c e l l number f o l l o w i n g 28 days o f a r t i f i c i a l l y induced c r y p t o r c h i d i s m . The p a r t i a l d e g e n e r a t i o n of the germinal e p i t h e l i u m observed i n one t u b u l e of a c o n t r o l t e s t i s  ( f i g . 110, 111),  cannot be e x p l a i n e d , but t h e form o f the degeneration i s of interest.  A c c o r d i n g t o Perey e t a l (1961), each stage o f the  c y c l e can be d i v i d e d i n t o substages i n which the component germ c e l l s develop i n d e p e n d e n t l y .  The d e g e n e r a t i o n o f o n l y  p a r t o f t h e c r o s s s e c t i o n s of t h i s t u b u l e c o u l d t h e r e f o r e r e p r e s e n t the l i m i t s of the substage comprising the p a r t i c u l a r stage o f t h e c y c l e i n v o l v e d .  I f t h i s i s t r u e , then i t  i s p o s s i b l e t h a t substages o f stages i n t h e s e m i n i f e r o u s c y c l e undergo independent testicular  atrophy.  degenerative r e a c t i o n s during  38  SUMMARY  1.  The a t r o p h i c r e a c t i o n o f t h e g e r m i n a l e p i t h elium that f o l l o w s l i g a t i o n of the vasa e f f e r e n t i a i n a d u l t mice and r a t t e s t e s was s t u d i e d a t each s t a g e o f the seminiferous  2.  cycle at various p o s t l i g a t i o n  periods.  The t i m i n g o f t h e c y c l e d i d n o t appear t o be d i s r u p t e d by t h e d e g e n e r a t i v e changes t h a t r e s u l t e d : a t a l l p o s t o p e r a t i v e p e r i o d s , t h e v i a b l e germ c e l l s were a s s o c i a t e d w i t h t h e same s t a g e s o f t h e c y c l e as they are i n the normal t e s t i s .  3.  The f r e q u e n c y w i t h which each s t a g e o f t h e c y c l e occurred  was measured i n t h e l i g a t e d t e s t i s o f  t h e mouse and found t o be unchanged from t h a t e s t a b l i s h e d in the control t e s t i s . 4.  The a t r o p h i c r e a c t i o n s a t each s t a g e o f t h e c y c l e f o l l o w e d a c o n s i s t e n t and w e l l - d e f i n e d sequence of e v e n t s .  These have been d e s c r i b e d  i n t h e form o f a  g e n e r a l scheme f o r t u b u l e d e g e n e r a t i o n .  The proposed  scheme c o n s i s t s o f 4 main s t e p s w h i c h , d u r i n g s t a g e s 1 IX o f t h e c y c l e , a r e : (1) t h e " s p e r m a t i d  sloughing"  39  stage,  (2)  t h e " m u l t i n u c l e a t e d body" s t a g e ,  " S e r t o l i c e l l c y t o p l a s m " s t a g e , and c e l l sloughing" stage.  (4)  (3)  the  the " S e r t o l i  D u r i n g s t a g e s X - X l l of t h e  c y c l e i n t h e mouse ( s t a g e s X - XIV i n t h e r a t ) ,  the  f i r s t 2 o f t h e 4 d e g e n e r a t i v e s t e p s d i f f e r and have been termed:  (1)  t h e " c e l l u l a r s l o u g h i n g " s t a g e , and  the "spermatocyte 5.  ghost c e l l "  (2)  stage.  Spermatogenesis was i n h i b i t e d from p r o c e e d i n g f u r t h e r t h a n pachytene spermatocytes  i n t u b u l e s which  were w e l l i n t o o r beyond t h e " m u l t i n u c l e a t e d body" s t a g e or t h e "spermatocyte  ghost c e l l " s t a g e of t u b u l e  degeneration. 6.  Four s t e p s i n t h e d e g e n e r a t i o n o f m u l t i n u c l e a t e d b o d i e s o f s p e r m a t i d s have been d i f f e r e n t i a t e d and described.  7.  C y t o p l a s m i c r e s i d u a l b o d i e s , sloughed by mature s p e r m a t i d s p r i o r t o s e v e r e t u b u l a r a t r o p h y , were o r g a n i z e d i n t o " r e s i d u a l body masses" w i t h i n t h e S e r t o l i cytoplasm.  cell  These r e s i d u a l body masses t h e n f o l l o w e d a  s i m i l a r l y s i s as m u l t i n u c l e a t e d b o d i e s . 8.  Degeneration  of seminiferous tubules a f f e c t e d  S e r t o l i c e l l s i n a d e f i n i t e manner:  the S e r t o l i  cell  40  n u c l e i i n c r e a s e d i n number, f r e q u e n t l y m i g r a t e d  away  from t h e basement membrane and f i n a l l y , d e c r e a s e d i n number as a r e s u l t o f S e r t o l i c e l l s l o u g h i n g i n t o t h e lumen.  41  LITERATURE CITED  Chowdhury, A.K., S t e i n b e r g e r , E., 1964 "A q u a n t i t a t i v e s t u d y of t h e e f f e c t o f h e a t on g e r m i n a l e p i t h e l i u m o f rat testes." Am. J . Anat., 115: 509 - 524. C l e g g , E . J . , M a c M i l l a n , E.W.,. 1965 "The uptake of v i t a l dyes and p a r t i c u l a t e m a t t e r by t h e S e r t o l i c e l l s of the rat t e s t i s . " J . A n a t . , Lond., 99.: 219 - 229. Clegg, E.J., 1963 " O b s e r v a t i o n s on t h e uptake of I n d i a ink by t h e s e m i n i f e r o u s t u b u l e s and e x c u r r e n t d u c t systems of t h e r a t t e s t e s . " J . Anat., Lond., 97: 154. Clegg, E.J., 1963 " S t u d i e s on a r t i f i c i a l c r y p t o r c h i d i s m : m o r p h o l o g i c a l and q u a n t i t a t i v e changes i n t h e S e r t o l i c e l l s of the r a t t e s t i s . " J. Endocrinol., 26: 567 - 574. Clegg, E.J., 1963 " S t u d i e s on a r t i f i c i a l c r y p t o r c h i d i s m : d e g e n e r a t i v e and r e g e n e r a t i v e changes i n t h e g e r m i n a l e p i t h e l i u m of t h e r a t t e s t i s . " J. Endocrinol., 27: 241 - 251. Clermont, Y., Harvey, S.C., 1965 " D u r a t i o n of t h e c y c l e of t h e s e m i n i f e r o u s e p i t h e l i u m o f n o r m a l , hypophysectomi z e d and hypophysectomized - hormone t r e a t e d a l b i n o rats." J . E n d o c r i n o l . , 76: 80 - 89. Clermont, Y., 1963 e l i u m i n man."  "The c y c l e of ,the s e m i n i f e r o u s Am. J . A n a t . , 112: 35.  epith-  C l e r m o n t , Y., L e b l o n d , C P . , 1959 " D i f f e r e n t i a t i o n and r e n e w a l o f spermatogonia i n t h e monkey, Macacus r h e o u s . " Am. J . Anat., 104: 237 - 273. Clermont, Y., P e r e y B,, 1957 "The s t a g e s of t h e c y c l e o f the seminiferous e p i t h e l i u m of the r a t : p r a c t i c a l d e f i n i t i o n s i n P A - S c h i f f - h e m a t o x y l i n and h e m a t o x y l i n -  42  e o s i n s t a i n e d s e c t i o n s . " Rev. Canad. B i o l . ,  16:  451 - 462.  Clermont, Y., P e r e y , B., 1957 " Q u a n t i t a t i v e study o f t h e c e l l p o p u l a t i o n o f t h e s e m i n i f e r o u s t u b u l e s i n immature rats." Am. J . Anat., 100: 241 - 267. Clermont, Y., M o r g e n t a l e r , H» 1955 " Q u a n t i t a t i v e study of spermatogenesis i n t h e hypophysectomized r a t . " E n d o c r i n o l o g y , 5J7: 369 - 382. ?  Clermont, Y., L e b l o n d , C P . , 1955 "Spermiogenesis o f man, monkey, ram and o t h e r mammals as shown b y t h e ' p e r i o d i c a c i d - S c h i f f technique." Am. J . Anat., 96.: 229 - 253. C l e r m o n t , Y., 1954 " C y c l e de l ' e p i t h e ' l i u m s e m i n a l e t mode de r e n o u v e l l e m e n t des spermatogonies chez l e hamster." Revue Canadienne de B i o l o g i e , 13_: 208 - 245. Clermont, Y., L e b l o n d , C P . , 1953 "Renewal o f spermatog o n i a i n t h e r a t . " Am. J . Anat., 93.: 475 - 501. Daoust, R., Clermont, Y., 1955 " D i s t r i b u t i o n of n u c l e i c a c i d s i n germ c e l l s d u r i n g t h e c y c l e o f t h e s e m i n i f e r ous e p i t h e l i u m i n t h e r a t . " Am. J . Anat., 96: 255 - 283.  D a v i s , J.R., F i r / l i t , C.F., 1966 "The g e r m i n a l e p i t h e l i u m of c r y p t o r c h i d t e s t e s e x p e r i m e n t a l l y i n d u c e d i n p r e p u b e r t a l and a d u l t r a t s . " F e r t i l and S t e r i l . , 17: 187 - 200.  D i e t e r t , S.E., 1966 " F i n e s t r u c t u r e o f t h e f o r m a t i o n and f a t e o f t h e r e s i d u a l b o d i e s o f mouse spermatozoa w i t h e v i d e n c e f o r t h e p a r t i c i p a t i o n o f lysosomes." J . Morph., 120:  317 - 346.  F i r l i t , C.F., D a v i s , J.R., 1965 "Morphogenesis o f t h e r e s i d u a l body o f t h e mouse t e s t i s . " Quart. J . M i c r . Sci., 106: 93 - 98. Lacy, p., L o f t s , B., 1965 " S t u d i e s on t h e s t r u c t u r e and f u n c t i o n o f mammalian t e s t i s . 1. C y t o l o g i c a l and  43  h i s t o c h e m i c a l observations a f t e r continuous treatment w i t h o e s t r o g e n i c hormone and t h e e f f e c t s of F.S.H. and L.H." P r o c . Roy. Soc. B., 162: 188 - 197. Lacy, D., 1962 function."  " C e r t a i n a s p e c t s of t e s t i s s t r u c t u r e B r i t . Med. B u l l . , 18: 205 - 208.  and  Lacy, D., 1960 " L i g h t and e l e c t r o n m i c r o s c o p y and i t s use i n t h e s t u d y of f a c t o r s i n f l u e n c i n g spermatogenesis i n the r a t . " J . Roy. M i c r . S o c , 79: 209 - 225. L e b l o n d , C P . , Clermont, Y., 1952 " D e f i n i t i o n of t h e s t a g e s of t h e c y c l e of t h e s e m i n i f e r o u s e p i t h e l i u m o f f t h e r a t . " Ann. N.Y. Acad. S c i . , 55: 548 - 573. Murphy, H.D., 1965 "Sertoli c e l l stimulation following i n t r a t e s t i c u l a r i n j e c t i o n s o f F.S.H. i n t h e hypophysectomized r a t . " P r o c , Soc. Exp. B i o l . Med., 118(4): 1202 - 1205. N e b e l , B.R., Murphy, C.J., 1960 "Damage and r e c o v e r y of mouse t e s t i s a f t e r lOOOr acute l o c a l i z e d X - i r r a d i a t i o n , with reference t o r e s t i t u t i o n c e l l s , S e r t o l i c e l l i n c r e a s e , and t y p e A s p e r m a t o g o n i a l r e c o v e r y . " Rad. Res., 12: 626 - 641. N i e m i , M., Kormano, M., 1965 " C y c l i c a l changes i n and s i g n i f i c a n c e o f l i p i d s and a c i d phosphatase a c t i v i t y i n t h e seminiferous t u b u l e s of the r a t t e s t i s . " A n a t . Rec., 151: 159 - 171. N i e m i , M. Kormano, M., 1965 "Response of t h e c y c l e o f t h e seminiferous e p i t h e l i u m of the r a t t e s t i s t o a r t i f i c i a l cryptorchidism." F e r t i l , and S t e r i l . , 16: 236 - 241. Oakberg, E.F., 1956(a) "A d e s c r i p t i o n of s p e r m i o g e n e s i s i n t h e mouse and i t s use i n a n a l y s i s o f t h e c y c l e of t h e s e m i n i f e r o u s e p i t h e l i u m and germ c e l l r e n e w a l . " Am. J . Anat., 9 9 ( 3 ) : 391 - 413. Oakberg, E.F. 1956(b) " D u r a t i o n of spermatogenesis i n t h e mouse and t i m i n g of s t a g e s o f t h e c y c l e ; o f t h e s e m i n i f erous e p i t h e l i u m . " Am. J . Anat., 99_: 507 - 516.  44  Oakberg, E.F., 1955(a) " S e n s i t i v i t y and t i m e o f degenera t i o n o f spermatogenic c e l l s i r r a d i a t e d i n v a r i o u s s t a g e s o f m a t u r a t i o n i n t h e mouse." Rad. Res., 2.: 369 - 391. Oakberg, of in J.  E.F., 1955(b) "Degeneration o f spermatogonia t h e mouse f o l l o w i n g exposure t o X - r a y s , and s t a g e s t h e m i t o t i c c y c l e a t which c e l l d e a t h o c c u r s . " Morph., 97.: 39 - 54.  O e t t l e , A.G., H a r r i s o n , R.G., 1952 " H i s t o l o g i c a l changes produced i n t h e r a t t e s t i s by temporary and permanent occlusion of the t e s t i c u l a r artery." J . Path. Bact., 64: 273 - 297. P e r e y , B., Clermont, Y., L e b l o n d , C P . , 1961 "The wave of t h e s e m i n i f e r o u s e p i t h e l i u m i n t h e r a t . " Am. J . of Anat., 108: 47 - 78. Reddy, K . J . , Svoboda, D.J., 1967 "Lysosomal a c t i v i t y i n S e r t o l i c e l l s o f normal and d e g e n e r a t i n g s e m i n i f e r o u s e p i t h e l i u m o f r a t t e s t e s . " Amer. J . P a t h . , 51: 1 - 17. Roosen-Runge, E.C., 1952 " K i n e t i c s o f spermatogenesis i n mammals." Ann. N.Y. Acad. S c i . , 55: 574 - 584. Roosen-Runge, E . C , 1951 " Q u a n t i t a t i v e s t u d i e s on s p e r matogenesis i n t h e a l b i n o r a t . 11. The d u r a t i o n o f spermatogenesis and some e f f e c t s o f c o l c h i c i n e . " Am. J . Anat., 88: 163 - 176. Roosen-Runge, E . C , G i e s e l , L.O., 1950 "Quantitative s t u d i e s on spermatogenesis i n t h e a l b i n o r a t . " Am. J . Anat., 87.: 1 - 30. S a p s f o r d , C.S., 1962 "Changes i n t h e c e l l s o f t h e sex c h o r d s and s e m i n i f e r o u s t u b u l e s d u r i n g t h e development of t h e t e s t i s o f t h e r a t and mouse." A u s t . J . Z o o l . , 10: 178-193. S a p s f o r d , C.S., 1962 "The development o f t h e t e s t i s o f t h e M e r i n o ram, w i t h s p e c i a l r e f e r e n c e t o t h e o r i g i n o f t h e a d u l t stem c e l l s . " A u s t . J . A g r i c . Res., 13.: 487 - 502.  45  Smith, G., 1962 "The e f f e c t s o f l i g a t i o n o f t h e v a s a e f f e r e n t i a and vasectomy on t e s t i c u l a r f u n c t i o n i n t h e adult r a t . " J . E n d o c r i n o l . , 23_: 385 - 399. Smith, B.V.K., Lacy, D., 1959 "Residual bodies of s e m i n i f e r o u s t u b u l e s o f t h e r a t . " N a t u r e , 184: 249 - 251. S t e i n b e r g e r , E., D i x o n , W.J., 1959 "Some o b s e r v a t i o n s on t h e e f f e c t o f h e a t on t h e t e s t i c u l a r g e r m i n a l e p i t h elium." F e r t i l . and S t e r i l . , 10: 578 - 595. Van Wagenen, G., 1926 "Degeneration and r e g e n e r a t i o n o f the seminiferous tubules a f t e r l i g a t i o n of the d u c t u l i e f f e r e n t e s i n t h e r a t . " Anat. Rec. 32: 225. Van Wagenen, G., 1925 "Changes i n t h e t e s t i s o f t h e r a t following l i g a t i o n of the ductuli e f f e r e n t i . " Anat. Rec, 29: 399. Van Wagenen, G., 1924 "Degeneration o f g e r m i n a l e p i t h e l i u m i n t h e t e s t i s o f t h e r a t as a r e s u l t o f e f f e r e n t d u c t ligation." A n a t . R e c , 27: 189.  4 6  KEY TO ABBREVIATIONS  AC acre-some ASP a t r o p h i c spermatids BM basement membrane DS d i v i d i n g p r i m a r y spermatocyte GC "ghost" c e l l HC head cap L s e m i n i f e r o u s t u b u l e lumen LC s p e r m a t i d s & c e l l u l a r d e b r i s i n lumen LS l e p t o t e n e p r i m a r y spermatocyte MF f u s i o n o f n u c l e a r membrane MNB m u l t i n u c l e a t e d body MNG m u l t i n u c l e a t e d "ghost" MS spermatid i n m i t o s i s NUC spermatid nucleus NN nucleolus PC pachytene p r i m a r y spermatocyte PS d i p l o t e n e p r i m a r y spermatocyte RB r e s i d u a l body RBM r e s i d u a l body mass RS r e s t i n g p r i m a r y spermatocyte SC S e r t o l i c e l l nucleus SCC S e r t o l i c e l l cytoplasm SCS S e r t o l i c e l l s sloughing SG " spermatogonium SP spermatid SS secondary spermatocyte V vacuole VF vacuole forming ZS zygotene p r i m a r y spermatocyte  PLATES  47 PLATE 1 The C y c l e of t h e S e m i n i f e r o u s E p i t h e l i u m i n t h e Mouse Fig. 2 - 1 3 . R e p r e s e n t a t i v e s e m i n i f e r o u s t u b u l e s from a n o r mal mouse t e s t i s showing t h e 12 s t a g e s o f t h e c y c l e o f the s e m i n i f e r o u s e p i t h e l i u m . The c l a s s i f i c a t i o n o f t h e c y c l e i s based on t h e f i r s t 12 s t e p s o f s p e r m i o g e n e s i s . A l l 16 s t e p s of s p e r m i o g e n e s i s ( 1 - 3 , t h e G o l g i phase; 4 - 7 , t h e cap phase; 8 - 12, t h e acrosome phase; and 13 - 16, t h e matura t i o n phase) are i d e n t i f i e d by t h e changes i n t h e a c r o s o m i c system o f s p e r m a t i d s as seen w i t h p e r i o d i c a c i d - S c h i f f . The h a e m a t o x y l i n s t e p has been o m i t t e d from t h e s t a i n i n g p r o c e d ure t o s i m p l i f y t h e changes i n t h e acrosomic system a t each stage of the c y c l e . The c y c l e i s a c o n t i n u o u s p r o c e s s o f germ c e l l r e n e w a l , w i t h no b e g i n n i n g o r end s t a g e s . The b e g i n n i n g of s p e r m a t o g e n e s i s , however, i s w i t h t h e m i t o s e s of t y p e A spermatogonia a t s t a g e IX o f t h e c y c l e ( f i g . 10; t a b l e 2 ) • The n u c l e u s and acrosome of stage IX s p e r m a t i d s undergo a marked e l o n g a t i o n , w h i c h c o n t i n u e s t h r o u g h stage X l l ( f i g . 13). A t l a t e stage X l l a new g e n e r a t i o n of s p e r m a t i d s app e a r s i n t h e g e r m i n a l e p i t h e l i u m and marks t h e b e g i n n i n g o f s t a g e 1 o f t h e c y c l e ( f i g . 2 ) . The o l d e r g e n e r a t i o n of s p e r m a t i d s b e g i n s t e p 13 o f s p e r m i o g e n e s i s . The o v e r l a p o f t h e two g e n e r a t i o n s o f s p e r m a t i d s p e r s i s t s t h r o u g h s t a g e s 1Vlll. Stage 11 ( f i g . 3) i s i n i t i a t e d by t h e appearance of p r o a c r o s o m i c g r a n u l e s , which e n l a r g e and f u s e i n t o one l a r g e g r a n u l e a t s t a g e 111 ( f i g . 4 ) . The head cap b e g i n s t o form at s t a g e IV ( f i g . 5) and extends over t h e n u c l e u s d u r i n g t h e n e x t two s t a g e s . A t s t a g e V l l ( f i g . 8 ) , t h e head cap c o v e r s over o n e - t h i r d of t h e n u c l e u s . T h e o l d e r g e n e r a t i o n o f s p e r m a t i d s a t t h e b e g i n n i n g of s t a g e V l l e n t e r step'16 of s p e r m i o g e n e s i s - t h e l a s t m o r p h o l o g i c a l s t e p of m a t u r a t i o n . D u r i n g s t a g e V l l l ( f i g . 9) t h e s e mature s p e r m a t i d s are r e l e a s e d i n t o t h e lumen o f t h e t u b u l e , l e a v i n g o n l y t h e one g e n e r a t i o n , step 8 spermatids i n the germinal e p i t h e l i u m . In f i g u r e 9, mature s p e r m a t i d s on t h e r i g h t s i d e of t h e t u b u l e have a l r e a d y been r e l e a s e d , b u t on t h e l e f t s i d e , most of t h e s p e r m a t i d s are s t i l l p r e s e n t . 725 X.  48  PLATE 2  F i g . 14. E l e c t r o n m i c r o g r a p h of a l o n g i t u d i n a l s e c t i o n of a s p e r m a t i d f r o m a c o n t r o l r a t t e s t i s showing t h e a n t e r i o r r e g i o n of t h e n u c l e u s (NUC), the acrosome (AC), and t h e dev e l o p i n g head cap (HC). The s p e r m a t i d i s i n a p p r o x i m a t e l y s t e p 8 of s p e r m i o g e n e s i s . Compare t h e morphology o f t h e head cap, which has been used as a c r i t e r i o n f o r the i d e n t i f i c a t i o n o f t h e stage of t h e s e m i n i f e r o u s c y c l e , w i t h t h a t o f s t e p 8 s p e r m a t i d s p r e p a r e d f o r l i g h t m i c r o s c o p y ( f i g . 9, and 57, 5 8 ) . The s u r r o u n d i n g S e r t o l i c e l l c y t o p l a s m (SCC), s e p a r a t e d from t h e s p e r m a t i d o n l y by t h e S e r t o l i plasma membrane, c o u l d p l a y an i m p o r t a n t r o l e n o t o n l y i n t h e t r a n s p o r t o f n u t r i e n t s from t h e basement membrane t o d e v e l o p i n g germ c e l l s , b u t a l s o i n t h e l o c a l r e g u l a t i o n of spermatog e n e s i s . The t i s s u e was f i x e d w i t h g l u t a r a l d e h y d e and osmium t e x r o x i d e . C o n t r a s t was improved w i t h u r a n y l a c e t a t e and lead c i t r a t e . 45,000 X.  49  PLATES 3,4,5  & 6  The a t r o p h i c r e a c t i o n a t each stage o f t h e semi n i f e r o u s c y c l e i n t h e mouse, f o l l o w i n g l i g a t i o n o f t h e v a s a e f f e r e n t i a i s r e p r e s e n t e d on p l a t e s 3, 4, 5, and 6 ( f i g . 15 8 6 ) . The d e g e n e r a t i v e changes i l l u s t r a t e d a r e summed up i n t a b l e 6, page 17. The g e r m i n a l e p i t h e l i u m i s o r i e n t a t e d i n a l l m i c r o g r a p h s w i t h t h e basement membrane a t t h e t o p and t h e lumen a t t h e bottom. Roman numerals r e p r e s e n t t h e 12 s t a g e s of t h e c y c l e . PAS - h a e m a t o x y l i n . 2000 X.  a  DAY  5  D A Y  "7  D A Y  a  DAY  5  DAY  7  DAY  50 PLATE 7  Sections of seminiferous tubules f o l l o w i n g l i g a t i o n of t h e v a s a e f f e r e n t i a , r e p r e s e n t a t i v e of t h e f i r s t 2 s t e p s o f t u b u l e degenera t i o n d u r i n g s t a g e s X - XIV of t h e seminiferous c y c l e i n the r a t . Iron haematoxylin. F i g . 87. Stage X l l of t h e s e m i n i f e r o u s c y c l e from a c o n t r o l r a t t e s t i s . The g e r m i n a l e p i t h e l i u m c o n s i s t s o f S e r t o l i c e l l n u c l e i (SC) and t y p e A spermatogonia (SG) a d j a c e n t t o t h e basement membrane; zygotene (ZS), t h e n pachytene (PC) primary spermatocytes; and s p e r m a t i d s (SP) i n s t e p 12 o f s p e r m i o g e n e s i s b e s i d e t h e lumen. 1350 X. F i g . 88. A p p r o x i m a t e l y s t a g e X l l of t h e s e m i n i f e r o u s c y c l e during the f i r s t step i n seminiferous tubule degeneration t h e " c e l l u l a r s l o u g h i n g " s t a g e . Spermatids have been p r e m a t u r e l y r e l e a s e d , and s e v e r a l pachytene p r i m a r y spermatoc y t e s (PC) a r e i n t h e p r o c e s s o f b e i n g sloughed i n t o t h e lumen ( L ) . Most zygotene (ZS), and some pachytene p r i m a r y s p e r m a t o c y t e s i n t h e g e r m i n a l e p i t h e l i u m s t i l l appear morp h o l o g i c a l l y n o r m a l . Some S e r t o l i c e l l n u c l e i (SC) have m i g r a t e d away from t h e basement membrane. 1250 X. F i g . 89 & 90. Regions o f 2 d i f f e r e n t s e m i n i f e r o u s t u b u l e s i n t h e "spermatocyte ghost c e l l " stage of t u b u l e degenera t i o n . I n f i g . 89, t h e number o f pachytene p r i m a r y s p e r matocytes i s reduced as a r e s u l t o f c e l l u l a r s l o u g h i n g and d e g e n e r a t i o n w i t h i n t h e g e r m i n a l e p i t h e l i u m . Two pachytene p r i m a r y spermatocytes w i t h p y k n o t i c n u c l e i have been sloughed i n t o t h e lumen; t h e remainder have e i t h e r p y k n o t i c n u c l e i o r have undergone c h r o m a t o l y s i s and appear as "ghost" c e l l s (GC) w i t h l i t t l e or no n u c l e a r m a t e r i a l and f a i n t PAS - p o s i t i v e c y t o p l a s m . Most zygotene spermatocytes s t i l l appear m o r p h o l o g i c a l l y n o r m a l . The c y t o p l a s m of t h e S e r t o l i c e l l s (SCC) i s much more c o n s p i c u o u s and c o n t a i n s numerous v a c u o l e s ( V ) . The S e r t o l i c e l l n u c l e i have i n c r e a s e d i n number, and a few are l o c a t e d away from t h e basement membrane. Towards t h e end o f t h e "spermatocyte ghost c e l l " stage ( f i g . 9 0 ) , as t h e degene r a t i o n o f t h e g e r m i n a l e p i t h e l i u m c o n t i n u e s , t h e number o f spermatocytes i s g r e a t l y d e p l e t e d . Numerous "ghost" c e l l s (GC) o f d e g e n e r a t i n g pachytene and zygotene spermatocytes are p r e s e n t . V a c u o l e s (V) i n t h e S e r t o l i c e l l c y t o p l a s m are abundant  1250  X  51 PLATE 8  Sections of seminiferous t u b u l e s r e p r e s e n t a t i v e of the steps of t u b u l e degeneration d u r i n g s t a g e s 1 - IX of t h e s e m i n i f e r o u s c y c l e i n the r a t . I r o n Haematoxylin. F i g . 91. Stage 111 o f t h e s e m i n i f e r o u s c y c l e d u r i n g t h e " s p e r m a t i d s l o u g h i n g " s t a g e . Most of s t e p 16 s p e r m a t i d s a r e s t i l l p r e s e n t i n t h e g e r m i n a l e p i t h e l i u m . A few s t e p 3 s p e r m a t i d s are about t o be s l o u g h e d . I n t e r m e d i a t e spermatogonia, pachytene p r i m a r y spermatocytes and S e r t o l i c e l l n u c l e i are m o r p h o l o g i c a l l y n o r m a l . 1450 X, F i g . 92, Approximately stage V l l of the c y c l e . During the l a t t e r p a r t of the "spermatid s l o u g h i n g " stage of t u b u l e d e g e n e r a t i o n . The younger g e n e r a t i o n o f s p e r m a t i d s have undergone p y k n o t i c changes, p r o d u c i n g n u c l e a r r i n g format i o n s o f c h r o m a t i n (ASP). The mature s p e r m a t i d s have a l l been e x f o l i a t e d . A few S e r t o l i c e l l n u c l e i have m i g r a t e d from t h e basement membrane. 1250 X. F i g . 93. A p p r o x i m a t e l y s t a g e V of t h e s e m i n i f e r o u s c y c l e a t t h e b e g i n n i n g o f t h e " m u l t i n u c l e a t e d body" s t a g e of tubule degeneration. M u l t i n u c l e a t e d bodies of spermatids (MNB) have formed as a r e s u l t of f u s i o n o f n u c l e a r memb r a n e s o f a d j a c e n t n e c r o t i c s p e r m a t i d s (ASP), The S e r t o l i c e l l c y t o p l a s m (SCC) becomes much more c o n s p i c u o u s and appears t o expand t o f i l l much o f t h e lumen ( L ) . S e r t o l i c e l l n u c l e i (SC) i n c r e a s e i n number and m i g r a t e away from t h e basement membrane. 1250 X. F i g . 94. The f u s i o n of 2 almost m o r p h o l o g i c a l l y normal spermatid n u c l e i at steps 4 - 5 i n spermiogenesis i n the mouse. The p o i n t of f u s i o n i s a t t h e o p p o s i t e end t o t h e head cap, b u t f u s i o n o f s p e r m a t i d n u c l e i i s g e n e r a l l y n o t r e s t r i c t e d t o any one p o i n t . PAS - h a e m a t o x y l i n . 10,300 X.  52  PLATE 9 F i g . 95. A l a t e r s t e p o f t h e " m u l t i n u c l e a t e d body" s t a g e •-'of t u b u l e d e g e n e r a t i o n than f i g u r e 94. Most o f t h e n e c r o t i c s p e r m a t i d n u c l e i have undergone membrane f u s i o n w i t h adjacent spermatid n u c l e i , forming l a r g e r m u l t i n u c l e a t e d b o d i e s (MNB). Note t h a t a l l t h e MNB a r e l o c a t e d i n t h e germinal epithelium. Iron haematoxylin. 1250 X. F i g . 96. A d e v i a t i o n from t h e u s u a l form o f t u b u l e degenera t i o n d u r i n g s t a g e s 1 - IX o f t h e c y c l e . The g e r m i n a l e p i t h e l i u m i s g r e a t l y r e d u c e d i n s i z e and t h e lumen (L) i s comp l e t e l y b l o c k e d w i t h germ c e l l s and c e l l u l a r d e b r i s ( L C ) . MNB a r e found i n t h e lumen as w e l l as i n t h e g e r m i n a l e p i t h e l i u m . The S e r t o l i c e l l n u c l e i have i n c r e a s e d i n number. Note t h e a c c u m u l a t i o n o f S e r t o l i c e l l n u c l e i a t a break i n t h e basement membrane a t t h e upper r i g h t c o r n e r . I r o n haematoxylin. 1250 X. F i g . 97. The end o f t h e " m u l t i n u c l e a t e d body" stage i n d e g e n e r a t i o n i s marked w i t h o n l y a few MNB r e m a i n i n g . The S e r t o l i c e l l c y t o p l a s m (SCC) i s r e l a t i v e l y c l e a r o f d e g e n e r a t i n g germ c e l l s and c o n t a i n s numerous v a c u o l e s ( V ) . I r o n haematoxylin. * 1200 X. F i g . 98. A s e c t i o n t a n g e n t i a l t o t h e lumen (L) d u r i n g t h e " S e r t o l i c e l l c y t o p l a s m " stage i n t u b u l e d e g e n e r a t i o n . The g e r m i n a l e p i t h e l i u m c o n s i s t s o f a few pachytene p r i m a r y s p e r m a t o c y t e s , spermatogonia and numerous S e r t o l i c e l l n u c l e i . The t a n g e n t i a l s e c t i o n i s an advantageous s e c t i o n t o i l l u s t r a t e t h e m i g r a t i o n o f S e r t o l i c e l l n u c l e i away from t h e b a s e ment membrane. I r o n h a e m a t o x y l i n , 1200 X.  53  PLATE 10 Seminiferous tubules i n the " S e r t o l i c e l l s l o u g h i n g " s t a g e o f s e m i n i f e r o u s t u b u l e de generation d u r i n g severe atrophy. F i g . 99. L o n g i t u d i n a l s e c t i o n of s e m i n i f e r o u s t u b u l e s from a r a t t e s t i s . The lower t u b u l e has undergone severe S e r t o l i c e l l s l o u g h i n g , r e s u l t i n g i n a t h i n germinal e p i t h e l i u m cons i s t i n g of S e r t o l i c e l l s , spermatogonia and a few p r i m a r y spermatocytes up t o t h e pachytene m a t u r a t i o n s t a g e . A mass of g e r m i n a l e p i t h e l i u m c o n t a i n i n g numerous S e r t o l i c e l l s (SCS) i s about t o be s l o u g h e d . I r o n h a e m a t o x y l i n . 550 X. F i g . 100. A higher m a g n i f i c a t i o n of the S e r t o l i c e l l sloughing c o n d i t i o n i n d i c a t e d i n f i g u r e 99 (SCS), b u t t a k e n from mouse t e s t i s . Numerous S e r t o l i c e l l n u c l e i (SC) and o c c a s i o n a l pachytene p r i m a r y spermatocytes (PC) a r e b e i n g sloughed i n t o t h e lumen. PAS - h a e m a t o x y l i n . 2400 X. F i g . 101. A group o f S e r t o l i c e l l s t h a t have been sloughed i n t o t h e lumen. The n u c l e i s t i l l appear m o r p h o l o g i c a l l y normal. A p o s s i b l e p y k n o t i c pachytene spermatocyte i s i n c l u d e d i n t h e S e r t o l i c e l l c y t o p l a s m i c mass. PAS - h a e m a t o x y l i n . 3600 X.  54  PLATE 11  The v a r i a b i l i t y i n t h e r a t e o f s e m i n i f e r o u s t u b u l e d e g e n e r a t i o n i n r a t t e s t e s t h a t have been s u b j e c t t o l i g a t i o n o f t h e v a s a e f f e r e n t i a f o r 4 days. Iron haematoxylin. F i g . 102. V a r i o u s degrees o f t u b u l e a t r o p h y i n d i f f e r e n t t u b u l e s . The d e g e n e r a t i v e changes make i t d i f f i c u l t t o a c c u r a t e l y determine t h e stage o f t h e c y c l e . The s t a g e s l i s t e d below a r e t h e r e f o r e a p p r o x i m a t i o n s . 500 X. Tubule B:  e a r l y , " m u l t i n u c l e a t e d body" stage o f t u b u l e d e g e n e r a t i o n d u r i n g s t a g e 111 of the c y c l e .  Tubule A:  more advanced " m u l t i n u c l e a t e d body" stage of degeneration d u r i n g stage V of t h e c y c l e .  Tubule B±i e a r l y "spermatocyte ghost c e l l " s t a g e of degeneration d u r i n g stage X of the cycle. Tubule C:  advanced " S e r t o l i c e l l s l o u g h i n g " stage of degeneration a t stage V l l of the c y c l e .  F i g . 103. An e x t r e m e l y a t r o p h i c s e m i n i f e r o u s t u b u l e (B, Bx, B 2 ) adjacent t o a tubule i n only a s l i g h t degenerative cond i t i o n (A, A J L , A 2 ) . Note t h a t a l t h o u g h t h e r a t e o f a t r o p h y v a r i e s i n d i f f e r e n t t u b u l e s , t h e degree o f d e g e n e r a t i o n g e n e r a l l y remains t h e same t h r o u g h o u t t h e l e n g t h o f t h e t u b u l e ( e g . t u b u l e B; and i n f i g u r e 102, t u b u l e s A and C ) . 500 X.  55  PLATE 12 The f a t e o f m u l t i n u c l e a t e d b o d i e s d u r i n g t h e " m u l t i n u c l e a t e d body" s t a g e of s e m i n i f e r o u s t u b u l e d e g e n e r a t i o n s t a g e s 1 - IX of t h e c y c l e i n the r a t t e s t i s . Iron haematoxylin. F i g . 104. Stage V I : t h e c e l l s l i n i n g t h e t u b u l e are t y p e B spermatogonia (SG), S e r t o l i c e l l n u c l e i (SC), and a few pachytene p r i m a r y spermatocytes (PC). Spermatids have formed m u l t i n u c l e a t e d b o d i e s (MNB) i n v a r i o u s s t a t e s of degenera t i o n . The MNB c o n s i s t s o f p e r i p h e r a l l y l o c a t e d s p e r m a t i d n u c l e i and a c e n t r a l c y t o p l a s m i c mass. Three s t a g e s o f MNB d e g e n e r a t i o n can be d i f f e r e n t i a t e d : Stage 1 MNB  c o n t a i n t h e most h y p e r c h r o m a t i c  nuclei.  Stage 2 MNB  show a marked r e d u c t i o n i n t h e i n t e n s i t y of t h e n u c l e a r and c y t o p l a s m i c s t a i n i n g .  Stage 3 MNB  c o n t a i n spermatids w i t h d i s r u p t e d nuclear membranes, and have been termed " m u l t i n u c l e a t e d g h o s t s " (MNG).  S e r t o l i c e l l n u c l e i (SC) are l o c a t e d c l o s e to the MNB and o f t e n conform t o t h e shape of t h e edge of a MNB. 1350 X. F i g . 105. A p p r o x i m a t e l y s t a g e V l l o f t h e c y c l e . Stages 1 and 2 MNB a r e p r e s e n t , and two l a t e s t a g e 3 MNG - which have become g r e a t l y d i s o r g a n i z e d . One MNG has been e x f o l i a t e d i n t o t h e t u b u l e lumen ( L ) . Stage 4 MNG i n v o l v e s t h e l y s i s of t h e r e m a i n i n g c e l l u l a r mass. The d i s a p p e a r a n c e of t h e MNG i s c o n c u r r e n t w i t h t h e f o r m a t i o n o f v a c u o l e s w i t h i n t h e germinal epithelium. 1350 X.  56 PLATE 13  The f a t e o f r e s i d u a l b o d i e s d u r i n g t e s t i c u l a r atrophy i n the r a t . Iron haematoxylin. F i g . 106. L o n g i t u d i n a l s e c t i o n o f a r e g i o n o f a normal semi n i f e r o u s t u b u l e i n s t a g e V l l . The l a r g e b a s o p h i l i c b o d i e s i n t h e lumen (L) and a t t h e edge o f t h e g e r m i n a l e p i t h e l i u m are c y t o p l a s m i c r e s i d u a l b o d i e s (RB) r e l e a s e d by s t e p 19 spermatids. 1250 X. F i g . 107. L o n g i t u d i n a l s e c t i o n o f a r e g i o n o f an a t r o p h i c s e m i n i f e r o u s t u b u l e i n s t a g e V l l . R e s i d u a l b o d i e s have been p h a g o c y t i z e d by S e r t o l i c e l l s and a r e b e i n g g a t h e r e d i n t o r e s i d u a l ' b o d y masses (RBM). M u l t i n u c l e a t e d b o d i e s (MNB) o f s t e p 7 s p e r m a t i d s can be seen i n v a r i o u s s t a t e s o f d e g e n e r a t i o n . S e r t o l i c e l l n u c l e i (SC) have i n c r e a s e d i n number, and some have m i g r a t e d away from t h e basement membrane. 1250 X. F i g . 108. Stage V l l : most r e s i d u a l b o d i e s have been o r g a n i z e d i n t o r e s i d u a l body masses and resemble t h e m u l t i n u c l e a t e d b o d i e s o f s p e r m a t i d s . R e s t i n g p r i m a r y spermatocytes (RS), S e r t o l i c e l l n u c l e i and a few pachytene p r i m a r y s p e r m a t o c y t e s l i n e t h e basement membrane. Numerous v a c u o l e s (V) a r e p r e s e n t w i t h i n t h e S e r t o l i c e l l c y t o p l a s m (SCC). 1250 X. F i g . 109. Cross s e c t i o n of a seminiferous t u b u l e s i m i l a r t o t h a t seen i n f i g u r e 108. Note t h e r e s i d u a l b o d i e s a r e l o c a t e d o n l y i n t h e r e s i d u a l body masses w i t h i n t h e c y t o p l a s m o f Sertoli cells. 1200 X.  57  PLATE 14  L i m i t e d a t r o p h y of a s e m i n i f e r o u s t u b u l e i n a c o n t r o l t e s t i s o f a mouse. PAS- haematoxylin• F i g . 110. Stage 1 o f t h e s e m i n i f e r o u s c y c l e : most of t h e t u b u l e appears normal, b u t a t t h e bottom o f t h e t u b u l e , b o t h g e n e r a t i o n s o f s p e r m a t i d s (SP) have undergone degene r a t i v e changes. Step 13 s p e r m a t i d s have been sloughed i n t o t h e lumen, and some o f t h e s t e p 1 s p e r m a t i d s have formed m u l t i n u c l e a t e d b o d i e s (MNB). 1300 X. F i g . 111. Stage X l l of t h e c y c l e : o n l y a s m a l l p o r t i o n of t h e t u b u l e appears n o r m a l . I n t h e a t r o p h i c r e g i o n of t h e t u b u l e , s t e p 12 s p e r m a t i d s have a l l been s l o u g h e d , and some secondary spermatocytes are i n t h e p r o c e s s o f b e i n g s l o u g h e d . M u l t i n u c l e a t e d b o d i e s (MNB) of secondary s p e r matocytes have formed i n a s i m i l a r manner as MNB o f s p e r m a t i d s . Some d i p l o t e n e p r i m a r y spermatocytes (PS) are p r e s e n t which have n o t y e t d i v i d e d t o form secondary spermatocytes. 1300 X. F i g . 112. H i g h e r m a g n i f i c a t i o n o f two secondary spermatoc y t e s whose n u c l e a r membranes have f u s e d t o g e t h e r . 2600 X.  58  PLATE 15 Changes i n t h e c o n t e n t s of t h e e p i d i d y m a l duct f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a i n mice* PAS - h a e m a t o x y l i n . 340 X. F i g . 113. testis.  S e c t i o n o f t h e c a p u t e p i d i d y m i d i s from a E v e r y d u c t s e c t i o n c o n t a i n s spermatozoa.  control  F i g . 114. A t 1 day p o s t l i g a t i o n , t h e c o n t e n t s of t h e e p i d i d y m a l d u c t show no apparent change. F i g . 115. A t 2 days p o s t l i g a t i o n , t h e number of i n t h e d u c t have d e c r e a s e d s l i g h t l y .  spermatozoa  F i g . 116. A t 4 days p o s t l i g a t i o n , some s e c t i o n s o f t h e d u c t are almost d e v o i d of spermatozoa. The r e g i o n of t h e d u c t t h a t has been c u t l o n g i t u d i n a l l y c o n t a i n s d i s o r g a n i z e d areas of n e c r o t i c spermatozoa and PAS - p o s i t i v e amorphous masses of mucus. F i g . 117. A t 5 days p o s t l i g a t i o n , t h e c o n t e n t s of a l l d u c t s e c t i o n s a r e abnormal. Many r e g i o n s of t h e d u c t a r e d e v o i d of spermatozoa. F i g . 118. A t 7 days p o s t l i g a t i o n , most r e g i o n s o f t h e d u c t are d e v o i d of spermatozoa o r c o n t a i n s m a l l clumps of nec r o t i c spermatozoa and mucus.  

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