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Ultrastructural and cytochemical study of mink (Mustela vison) spermatozoa Kim, Jong-Wook 1976

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AN ULTRASTRUCTURAL AND CYTOCHEMICAL STUDY OF MINK  (MUSTELA VISON)  SPERMATOZOA  by JONG-WOOK KIM B . S c . , Chung-Buk U n i v e r s i t y , M.Sc,  A THESIS THE  Tohoku  University,  Korea, Japan,  1956 1970  SUBMITTED IN PARTIAL FULFILLMENT OF REQUIREMENTS DOCTOR OF in  FOR THE DEGREE OF PHILOSOPHY  t h e Department of  ANIMAL  We  accept to  THE  this  SCIENCE  thesis  the required  as c o n f o r m i n g standard  UNIVERSITY OF BRITISH  COLUMBIA  December, 1976  @  Jong-Wook Kim, 1976  In p r e s e n t i n g t h i s  thesis  an advanced degree at the I  Library shall  f u r t h e r agree  for  fulfilment of  the requirements f o r  the U n i v e r s i t y of B r i t i s h Columbia,  make i t  freely available  that permission  for  I agree  r e f e r e n c e and  f o r e x t e n s i v e copying o f  this  that  study. thesis  s c h o l a r l y purposes may be granted by the Head of my Department or  by h i s of  in p a r t i a l  this  written  representatives. thesis  i s understood that  f o r f i n a n c i a l gain s h a l l  permission.  Department of  Animal Science  The U n i v e r s i t y of B r i t i s h 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  It  Columbia  copying or p u b l i c a t i o n  not be allowed without my  ii ABSTRACT  This  s t u d y was  structure,  cytochemistry  spermatozoa which their  relevance Mature  and m a t u r a t i o n c h a n g e s  to a r t i f i c i a l  mating.  from  research  i n this  from the t e s t i s  the vagina  Conventionally  a transmission  cytochemically The mink  and  o f f e m a l e mink  prepared thin  electron  localized  epididymis  microscope.  Enzymes  head  showed  anterior  border of the e q u a t o r i a l  and  one a t t h e p o s t a c r o s o m a l s h e a t h on e a c h  two c o n n e c t e d h u m p - l i k e  show a s t r o n g  acid  t o be a s p e c i e s - s p e c i f i c  attachment  increased  (p < 0.01)  the r e p r o d u c t i v e Although significantly reproductive significantly was  during  structures at  side.  phosphatase  structural  acrosome,  These  activity,  feature  which  o f t h e ovum o r f o r  in fertilization.  the p o s t a c r o s o m a l s w e l l i n g  on t h e  segment o f t h e  m i g h t be n e c e s s a r y f o r t h e r e c o g n i t i o n sperm-ovum  were  s i x swellings  the  appeared  after  i n spermatozoa.  spermatozoon  which  also  s e c t i o n s were o b s e r v e d  aspects:  swellings,  and i n  study.  immediately  dorsoventral  length  o f mink  insemination.  d a r k mink were u s e d  were r e l e a s e d  ultra-  c o r p u s and c a u d a ) o f t h e male mink, and were  collected  under  the  are important i n b i o l o g i c a l  standard  Spermatozoa (caput,  undertaken to i n v e s t i g a t e  The o c c u r r e n c e o f  i n s p e r m a t o z o a was  significantly  the passage of spermatozoa  through  tract.  the t o t a l during tract,  length  o f t h e head  d i d not change  t h e p a s s a g e o f s p e r m a t o z o a down t h e the a n t e r i o r  acrosomal length  d e c r e a s e d (p < 0.001),  significantly  increased  while  (p <  was  the p o s t a c r o s o m a l  0.05).  The  cell  acrosome,  membrane on t h e p e r i p h e r a l  with the exception  significantly spermatozoa The  five  then  appeared  separated minor  joining  capitulum  the passage o f  continuous but  w h i c h was f o l l o w e d  fibers.of  were f o u n d  was  tract.  forming at f i r s t  w i t h the dense  Some axoneme r e m n a n t s column  (p < 0.05) d u r i n g  t o show d o r s o v e n t r a l l y  columns,  of the  o f t h e t i p o f the acrosome,  through the r e p r o d u c t i v e  neck  laterally and  separated  part  by two m a j o r  a striated  the a x i a l  i n the i n t e r i o r  r i n g and  fiber  bundle.  of the  bundle.  The s h a p e o f t h e a n n u l u s was t r i a n g u l a r sections.  in  The o c c u r r e n c e o f t h e c y t o p l a s m i c  significantly  decreased  (p < 0.001) d u r i n g  longitudinal  droplet  was  the passage of  spermatozoa  t h r o u g h t h e t e s t i s and e p i d i d y m i s . The m o t i l i t y o f '  spermatozoa  was s i g n i f i c a n t l y  spermatozoa  passed the s u c c e s s i v e  increased  (p < 0.05) a s  parts  of the r e p r o d u c t i v e  tract. The  a c t i v i t i e s of acid  and a l k a l i n e  p h o s p h a t a s e s , ADPase,  A T P a s e and DOPA o x i d a s e were f o u n d t o be d i s t r i b u t e d head,  m i d d l e and p r i n c i p a l  Glucose-6-phosphatase,  activities  while the e s t e r a s e  extended  t o t h e head  base.  enhanced  the passage  t o be c o n f i n e d  to the  dehydrogenase  The a c t i v i t y  was n o t d e t e c t e d .  most enzyme a c t i v i t i e s  during  esterase,  d e h y d r o g e n a s e s , and  and m a l a t e  6-phosphogluconic dehydrogenase Although  non-specific  and i s o c i t r a t e  d i a p h o r a s e a c t i v i t i e s were s e e n  middle piece,  of  of epididymal spermatozoa.  5-nucleotidase,  malate, s u c c i n a t e , l a c t a t e NADH  pieces  i n the  o f spermatozoa  of spermatozoa  were  through the  reproductive alkaline  tract,  s e v e r a l enzyme  p h o s p h a t a s e s , ADPase, A T P a s e ,  n a s e ) were d i s t i n c t l y r e d u c e d semen r e c o v e r e d  from  p r e s e n c e o f enzyme or  activities  inhibiting  female r e p r o d u c t i v e  tract  and m a l a t e  i n spermatozoa  f e m a l e mink  (acid  from  and dehydroge-  ejaculated  f o l l o w i n g mating.  f a c t o r s i n the seminal was  discussed.  The plasma  V  TABLE  OF  CONTENTS Page  ABSTRACT  i i  TABLE OF CONTENTS  .  v  L I S T OF TABLES  ix  L I S T OF FIGURES  x  ACKNOWLEDGEMENTS  xi  GENERAL  INTRODUCTION  CHAPTER  I.  1  AN ULTRASTRUCTURAL STUDY SPERMATOZOA  A.  Introduction  B.  Review  OF MINK 3 3  of r e l a t e d studies  literature  4  of spermatozoa  4  1.  Early  2.  General  3.  The s p e r m a t o z o a n  morphology  o f mink  spermatozoa  . .  head  5 6  a.  The n u c l e u s  6  b.  The a c r o s o m e  6  c.  The p e r f o r a t o r i u m  8  d.  The p o s t a c r o s o m a l s h e a t h  9  e.  The c e l l  9  membrane  4.  The S p e r m a t o z o a n  neck  10  5.  The spermatozoan  tail  13  a.  The a x i a l  fiber  bundle  b.  The m i t o c h o n d r i a l  c.  The a n n u l u s centriole )  13  sheath  16  (Jensen's r i n g ,  ring 17  d.  The c y t o p l a s m i c  e.  The f i b r o u s  f.  The end p i e c e  droplet  18  sheath (terminal  and methods  19 piece)  . . . .  19  C.  Materials  20  D.  Results  21  1.  The s p e r m a t o z o a n head  21  2.  The s p e r m a t o z o a n neck  23  vi Page 3. E.  F. CHAPTER  The s p e r m a t o z o a n  tail  24  Discussion  36  1.  The s p e r m a t o z o a n head  36  2.  The s p e r m a t o z o a n neck  39  3.  The spermatozoan  41  tail  Summary  44  I I . CYTOCHEMICAL  OBSERVATIONS  OF MINK  SPERMATOZOA  46  A.  Introduction  46  B.  Review o f r e l a t e d l i t e r a t u r e 1. E a r l y s t u d i e s of spermatozoan c y t o c h e m i s t r y 2. The r o l e o f enzymes i n t h e s p e r m a t o z o a n motility  48 48 49  3.  The r o l e  51  4.  E p i d i d y m a l s p e r m a t o z o a as a model enzyme s t u d y  o f enzymes i n f e r t i l i z a t i o n  . . .  f o r the 53  Co  M a t e r i a l s and m e t h o d s  56  D.  Results  58  1.  Phosphatases  58  2.  Esterase  58  3.  Dehydrogenases  E.  F. CHAPTER  and o x i d a s e  59  Discussion  63  1.  Phosphatases  63  2.  Esterase  70  3.  Dehydrogenases  and o x i d a s e  72  Summary III.  78  ULTRASTRUCTURAL AND CYTOCHEMICAL CHANCES OF MINK SPERMATOZOA COLLECTED FROM THE REPRODUCTIVE TRACT AND SEMEN  80  A.  Introduction  80  B.  Review  82  of r e l a t e d l i t e r a t u r e  1.  Early studies  of the r e p r o d u c t i v e  2.  Morphological  changes  .  tract  . .  82 83  vii Page  3.  4.  C.  The  cytoplasmic  b.  The  acrosome  84  c.  The  cell  85  d.  The  middle piece  e.  Abnormal  spermatozoa  87  f.  Specific  gravity  87  g.  Reflecting capacity  2. 3.  droplet  83  membrane  87  to the l i g h t  . . .  88  Chemical changes  88  a.  Chemical composition  b.  Enzyme d i s t r i b u t i o n  89  c.  Metabolism  89  . . . . . . . . .  88  P h y s i o l o g i c a l changes  90  a.  Fertility  90  b.  Motility  92  c.  E o s i n o p h i l i c property  94  d.  Temperature  94  e.  Resistance  f.  Net  g.  Agglutinating property  95  h.  Contact  .96  Materials 1.  D.  a.  shock to a l k a l i n i t y  negative  surface  acidity  .  charge  and methods  The p r e p a r a t i o n v i a b i l i t y test  95 95  ability  97  The p r e p a r a t i o n o f m a t e r i a l reproductive tract  The  and  from  the 97  of e j a c u l a t e d  semen .and 97  procedure f o r e l e c t r o n microscopy . . . .  Results  99  . . 101  1.  The  viability  of spermatozoa  2.  Changes  3.  Changes i n t h e morphology spermatozoan head  i n the spermatozoan  101 head  length  . .  101  of the 106  a.  The  acrosome  b.  The  cell  c.  Spermatism  107  d.  The  109  membrane  swelling  . 106 107  viii Page 4. 5. E.  F.  Changes i n the morphology spermatozoan t a i l Changes  of the 109  i n t h e enzyme a c t i v i t y  . . . . . . .  110  Discussion  121  1.  The  121  2.  Changes  3.  Changes i n t h e morphology spermatozoan head  v i a b i l i t y of spermatozoa i n the spermatozoan  length  . .  124  of the  4.  Movements o f t h e c y t o p l a s m i c  5.  Changes  Summary  head  125 droplet  i n t h e enzyme a c t i v i t y  . . .  127 128 132  GENERAL SUMMARY  134  LITERATURE  138  CITED  ix L I S T OF  No.  TABLES  Title  Page  1  The m e t h o d s o f enzyme  2  The p e r c e n t o f m o t i l i l e s p e r m a t o z o a and u n s t a i n e d spermatozoa c o l l e c t e d from s u c c e s s i v e p a r t s of " t h e r e p r o d u c t i v e t r a c t and e j a c u l a t e d semen . . . .  102  D i m e n s i o n a l changes s p e r m a t o z o a n head  104  3 4  5  localization  at different  portions  57  of the  C h a n g e s i n t h e c e l l membrane c o n f i g u r a t i o n and i n t h e o c c u r r e n c e o f s w e l l i n g s and t h e c y t o c h e m i c a l droplet  108  C h a n g e s i n enzyme a c t i v i t i e s l o c a l i z e d i n t h e s p e r m a t o z o a f r o m t e s t i s , e p i d i d y m i s and e j a c u l a t e d semen °  H i  X  L I S T OF  No. 1-4  FIGURES  Title Ultrastructure  Page  of the epididyrnal  spermatozoan  head  28  5-8  Ultrastructure  of the spermatozoan  9-13  Ultrastructure  o f t h e neck  in  and  neck  middle  . . . .  30  piece  spermatozoa  32  14 - 23  Ultrastructure  . . . .  35  24 - 37 38  Enzyme l o c a l i z a t i o n i n e p i d i d y r n a l s p e r m a t o z o a S c h e m a t i c d r a w i n g o f a mink r e p r o d u c t ive t r a c t t o show t h e p o s i t i o n o f t h e v a r i o u s p a r t s (shaded r e g i o n s ) The p o s i t i o n s o f t h e head m e a s u r e d  62  98 100  A c r o s o m a l c h a n g e s o f s p e r m a t o z o a n heads d u r i n g passage through the r e p r o d u c t i v e tract and s u b s e q u e n t e j a c u l a t i o n ( s a g i t t a l s e c t i o n s )  114  S a g i t t a l s e c t i o n s o f t h e s p e r m a t i d s and spermatozoa i n the t e s t i s  116  B i z a r r e f o r m s o f t h e a c r o s o m e and t h e s e p a r a t i o n o f t h e acrosome f r o m t h e n u c l e u s o f t h e head i n t e s t i c u l a r and c a p u t spermatozoa ( s a g i t t a l s e c t i o n s )  118  The movement o f t h e c y t o p l a s m i c d r o p l e t , t h e a d h e s i v e n e s s o f t h e c e l l membrane t o t h e t i p o f t h e a c r o s o m e , and t h e o c c u r r e n c e o f t h e satellite fibrils . .  120  39 40 - 44  45 - 49 50 - 54  55-58  of the spermatozoan  tail  AC KNOWLE DGEME NTS I would Dr.  W.D.  like  Kitts,  Agricultural Sincere  thanks  professors Dr. Mrs.  this  Dr. C R .  M.A. T u n g  f o r assistance  during  and e n c o u r a g e m e n t .  the design  opportunity  advice  of the  my t h a n k s t o  D r . R.G. P e t e r s o n  and g u i d a n c e ,  M r s . P.K. G i l l  assistance,  and p r o g r e s s  to record  Krishnamurti,  f o r their  M. S t r i k e r ,  technical  and t h e Dean o f t h e F a c u l t y o f  a r e due t o D r . M.S. Ahmad f o r h i s i n v a l u a b l e  and h e l p  I take  o f a l l t o e x p r e s s my g r a t i t u d e t o  my s u p e r v i s o r  Sciences,  suggestions study.  first  and  t o Mr. G.  and Mr. J . C o l e  Galzy,  f o r their  and t o M r s . G. H u c h e l e g a and  M i s s F . E . Newsome f o r t h e i r  generous  help  on numerous  occasions. I would  also  o f Chung-Buk Dr.  H.K. Song  encouraging This  like  t o t h a n k D r . K.H. -Yeon, t h e P r e s i d e n t  University  i n Korea,  Professor  f o r their  continued  efforts  my p r o g r e s s  research  through  was s u p p o r t e d  RB-1974-2) a n d by t h e N a t i o n a l (A-132). from  Seoul  The A s i a F o u n d a t i o n t o Vancouver  organizations  the past  I extend  i n h e l p i n g and several  years.  by A g r i c u l t u r e C a n a d a  Research C o u n c i l provided  f o r the author. my  H . J . L e e and  gratitude.  a travel  (EMR  o f Canada grant  To a l l t h e s e  1  GENERAL  Following studies  practical  the  artificial It  have  the a p p l i c a t i o n  for  and  the i n v e n t i o n  of spermatozoa  However,  made by many  o f the b a s i c  insemination known t h a t  F o r example,  abnormal  occurrence  are  spermatozoan  there  spermatozoa  Onstad,  1967).  food  ratio,  color  reduction  to predict  ( E n d e r s , 1952;  o f t h e mink  but a l u x u r i o u s  b r e e d i n g season which  only  a small  amount  geographical,  hinder  elaborate  However, imminent.  with f e r t i l i t y i n  pattern,  t i m e , and enzyme  few r e p o r t s  i s confined  the a p p l i c a t i o n  organized,  are available 1965;  to the northern  t h e mink p r o d u c e s  f u r . Furthermore,  o f mink  about  neither••  t h e mink has  one month  of the y e a r ,  per e j a c u l a t i o n . factors  tend  spermatozoa.  o f A I i n t h e mink  a r e becoming much l a b o r  activity  on t h e s p e r m a t o z o a o f  Ishikawa e t a l . ,  lasts  methy-  fertility.  e c o n o m i c a l , and p h y s i o l o g i c a l  As mink f a r m s  professionally  level,  o f semen c a n be a c q u i r e d  studies  live-dead  t h e form o f t h e acrosome, t h e  a r e numerous r e p o r t s  distribution  nor wool,  These  correlate  swimming  of the Northern Hemisphere;  short  succeeded with  physiological, morphological  domestic animals, comparatively  on mink  parts  (1907)  field  (AI) of domestic a n i m a l s . several  as i n d i c a t o r s  Although  The  in this  o f d y n e i n arms i n an u l t r a s t r u c t u r a l  utilized  other  fundamental  investigators.  knowledge  when I v a n o v  motility,  lene blue or r e s a z u r i n  to  been  chemical a s p e c t s of spermatozoa  ratio,  and  of the microscope,  purposes began  i s well  mammals.  a  INTRODUCTION  larger  industry i s  and more  i s required  d u r i n g the  2 short  breeding  about  10  per  genitalia, et  season.  cent'of  different  Application these  while  pairing.  partners  o f AI  particular  general  m a l e mink  notable suffer lack  Pairing  apparently  to the  mink  problems  advantages  of AI:  to  diseases.  attempted to  f o r 30  insufficient  hand, b e c a u s e excised tozoa  of  et  genetic  the  of  present  the  ease  maturation  facts  that  anomalies of  the  (Ishikawa  occurs  of  times  fertility.  only  eliminates  a l s o ensures  the  improvement, d e c r e a s i n g purposes  i n mink has success  has  (Adams,  recovering  at  the  end  of  h a v e been u s e d  Adams, 1975;  and  been  control  1975).  the of  sporadically  been a c h i e v e d On  the  spermatozoa  Ahmad e t  the  due  other  from  i n AI  breeding o f mink  the  aspects  of  has  studied  the  (Pomytko  spermatozoa,  ultrastructural  epididyrnal spermatozoa,  spermatozoa.  season,  a l . , 1975b).  t o o b t a i n b a s i c knov/ledge o f mink  o f mink  several  and t h e p o s s i b l e u s a g e o f t h e p r e s e r v e d s p e r m a -  investigation  cytochemical  AI  b a s i c knowledge  spermatozoa  In o r d e r  the  libido  i n d u s t r y not  little  p e l t e d male mink  a l . , 1972;  of  i n t h e mink b u t  Although  years,  epididymis  epididyrnal  from  increase  number o f m a l e s r e q u i r e d f o r b r e e d i n g venereal  are  A l s o , a c o n s i d e r a b l e number o f t h e mink a r e i n j u r e d  to f i g h t i n g  with  the  c o p u l a t i o n p r o b l e m s and  a l . , 1965 ) .  due  Especially  as  well  as  and the  3 Chapter AN A.  shortly 1646),  Ham  first  after  the  physiology  first  with  recently Ruska,  observed  invention  numerous s t u d i e s  appreciation  of  of  the  with  the  the  of  spermatozoa.  The  been e l u c i d a t e d  and  extensively  the  by  the  species.  Although  structure  of  to  the  several  there  mammalian  undertaken and  the  the  light  i n the  mink  of  microscope.  same b a s i c  i s no  present  the  s i g n i f i c a n c e of mink.  also  techniques. been Although there  among  the  the  ultraof  a  investigation  to d i s c u s s  the  have  record  fine structure^of  of  the  of  fine structure  The  scanning  structure,  on  and  thin  have  the  physiology  and  spermatozoa  species  there  more  (Knoll  structure  to d e s c r i b e  reproductive  and  have e n r i c h e d  numerous s t u d i e s  spermatozoa.  accomplished  histochemical  electron  spermatozoa,  been  embedding,  structure  Detailed  transmission  external  s h a p e and  are  has  ago,  (Kircher,  microscope  techniques  have the  years  the  microscope  electron  mammalian  the  size,  300  microscope  structure  a p p l i c a t i o n of  using  almost  knowledge of  in fixation,  spermatozoa  study d e s c r i b i n g  basic  i n t e r n a l and  a v a r i a t i o n i n the  spermatozoa  of  light  p h y s i o l o g i c a l aspect  studied  mammalian  of  cleaving  Spermatozoa from  was  SPERMATOZOA  have b e e n r e c o r d e d .  improvement  freeze  understanding  the  spermatozoan  advent  The  and  of  spermatozoa  microscope,  sectioning  is  MINK  spermatozoa  i n the  improvement  1932).  electron  all  STUDY OF  INTRODUCTION  Since  and  ULTRASTRUCTURAL  I  mink  this  structure  4 B.  REVIEW OF  1.  Early  RELATED  Studies  of  LITERATURE Spermatozoa  S p e r m a t o z o a were f i r s t 1938)  soon a f t e r  Almost  150  o b s e r v e d by Ham  the i n v e n t i o n  years  later,  Eventually, production  the t e s t i s (Peltier,  was  and Dumas  the s e x u a l l y  p r o v e n as  1835).  The egg  spermatozoon  (Schweigger-Seidel,  single  and e q u a l  cells  individual strated that of  the j o i n i n g  of the egg,  somatic c e l l During  employed  van  early  end  piece.  new  (1883)  demonwith  techniques  and  interpretations  Even  notions  (1902) d e s c r i b e d a neck  be  1865;  century,  became more Leydig,  a spermatozoon  and a t a i l  of a connecting  accurate  1883;  Jensen,  as an o r g a n i s m  w h i c h was piece,  tracts  (Hartsoeker,  t h r o u g h o u t the 19th  Schweigger-Seidel,  consisting  observations  o f what s h o u l d  reported  of spermatozoan morphology  o f a head,  of  the p r e s e n c e of a l i m e n t a r y  improved  three parts  as  t h e m i c r o s c o p e and  by p r e c o n c e i v e d  As o p t i c s  consisting  to the  and  t h e number  1694).  Retzius  1861)  half  i n s p e r m a t o z o a was  1887).  spermatozoan  of the spermatozoon  and m i c r o - e m b r y o s  1845;  of  were r e c o g n i z e d  each c o n t a i n e d  period,  i n a germ c e l l .  (Donne,  male.  (Gegenbauer,  Beneden  that  chromosomes.  were p r i m i t i v e  descriptions  potent  contribution  1875).  that  ( 1 8 2 4 c ) showed  the s i t e  1865)  of p r o n u c l e i  showing  this  were i n f l u e n c e d present  in their  ( v a n Beneden,  (Meyer,  of the l i g h t m i c r o s c o p e .  Prevost  s p e r m a t o z o a were p r o d u c e d by  i n 1677  main  divided piece,  into and  5 Through  the development  (polarising,  ultraviolet,  ference-contrast,  and  and  century,  the knowledge  Although  the e a r l y  (Seymour  Benmosche,  1954,  1955;  and  scope,  made p o s s i b l e  spermatozoa  1957a,b,  1966;  2.  described  general,  namely  and end The  1955,  by  1962,  rapidly. with  the  information Wu  and  the i n t r o d u c t i o n  t h e improvement combined  of  the micro-  ultrastructural  Anberg,  1970,  Saacke  of  embedding,  with  i n the e l e c t r o n  1959;  1965;  o f Mink  spermatozoon  features  the 20th  has grown  1952;  1955),  and o f  1957;  1975;  study  Hancock,  Blom  and  and A l m q u i s t ,  1964a,  1972a,b,c).  General Morphology mink  during  new  a more d e t a i l e d  1961,  P e d e r s e n , 1970a,bj  Mature  Schnall,  techniques  F a w c e t t , 1958, 1960,  limited  resolution  (Afzelius,  Birch-Andersen, b;  only  t e c h n i q u e and  post-staining  inter-  of histochemistry  techniques  Bradfield,  of increased  of  phase-contrast,  microscope observations  1941;  achievement have  of microscopes  of the spermatozoon  electron  thin-sectioning  fixation,  analytical  t e c h n i q u e added  and  McKenzie, the  chemical  cell"  electron,  kinds  fluorescence),  physical  "whole  o f new  Retzius  the head,  Spermatozoa has  the b a s i c  (1902)  morphological  f o r the spermatozoon i n  neck, m i d d l e p i e c e ,  principal  piece,  piece. overall  length  o f mink  spermatozoon  The  head  a p p e a r s a l m o s t r o u n d when s e e n  the  b r e a d t h i s a p p r o x i m a t e l y t h e same as  7.5  um).  It i s flat  and  about  1 um  i s about  from the f l a t  thick  the l e n g t h (Onstad,  70  um.  side;  (about 1967).  6 3.  The S p e r m a t o z o a n The  which  Head  spermatozoan head  i s covered  consists  at the a n t e r i o r  mainly  of the nucleus  by t h e a c r o s o m e and a t t h e  posterior  by t h e p o s t a c r o s o m a l  sheath.  envelopes  the e n t i r e  the n u c l e a r c o v e r i n g s .  a.  The N u c l e u s .  the nucleus  increasing  over  The g r e a t e r p a r t  filled  homogeneously  cell  with  concentration posteriorly  s u r f a c e of the nucleus  fossa,  f o r the attachment  The  1952),  represent  which  (Mann, b.  1936).  The  i s variable  1970). (Retzius,  Fawcett  The n u c l e u s  1902;  b y a membrane  (1958) s u g g e s t e d process  and may  o f metabo-  that  of chromatin  they during  c o n t a i n s a s m a l l amount o f n o n -  ( B i s h o p and W a l t o n ,  1966a) and p o l y s a c c h a r i d e  1964).  The A c r o s o m e .  the acrosome Although covering, ture  slightly  o f gaseous end-products  due t o a c o n d e n s a t i o n  protein  which i s  of the c a p i t u l u m of the connecting  are not l i m i t e d  1952).  spermiogenesis. basic  (Friend,  contains vacuoles  an a c c u m u l a t i o n  (Schnall,  appeared  often  i s o c c u p i e d by  has a r e c e s s , t h e i m p l a n t a t i o n  to species (Fawcett, nucleus  Schnall,  lism  with  The s h a p e o f t h e i m p l a n t a t i o n f o s s a  according  membrane  chromatin  i n the nucleus  caudal  piece.  o f t h e head  d e n s e l y packed  distributed  The c e l l  A secretion  (Burgos  Hancock  and F a w c e t t ,  apparatus  1955; 'Anberg,  (1957a) p o s t u l a t e d a d o u b l e  t h e acrosome  closely  of the Golgi  applied  i s a membrane-limited  to the tapering  anterior  forms  1957).  acrosomeal cap-like  struc-  portion  of the  7 nucleus  (Fawcett, 1958).  occupied density  by  mature  acrosome Fawcett  by  beyond  1970).  i t s three  the a n t e r i o r  segment e x t e n d i n g back a differentiated  portion  (Fawcett,  segment  1970; varies  Bane,  has  a species-specific The  1966)  galea  structure  (Fawcett, The  and  margin  electron  distinguished  segment a  of the  comprising The  pro-  main nucleus;  the  shape  caudal  of  the  i s a c h a r a c t e r i s t i c of the s p e c i e s 1966a);  structural  feature  cap which  the acrosome  the  among s p e c i e s  the acrosome-postacrosomal  i s an  portion  (Nicander  sheath  (Koehler,  artifactual  junction  1969).  seems t o be  o f the c e l l  equa-  a separate  loosening membrane  1975). i s composed  of carbohydrate, l i p i d ,  ( C l e r m o n t e_t a_l. , 1955b;  1965).  Moreover,  the acrosome  (Teichman  The  spermatozoa Williams,  half  1957a).  i n i t s length  o r head  1966a;  the nucleus;  segment  (Hancock,  the  c a n be  apical  the a n t e r i o r  protein  1970).  an  of  of  and Walton',  acrosome  segments:  of the a n t e r i o r  acrosome  structure  shape  B i s h o p and W a l t o n ,  capitis  overlying  elevation  The  of the acrosome  1965,  and  (Bishop  equatorial  and  and  over  o f t h e acrosome  a p i c a l ' segment  the s i z e  among s p e c i e s  Phillips,  appearance  torial  t h e membranes i s  o f homogeneous s u b s t a n c e o f m o d e r a t e  spermatozoa,  differs and  jecting  and  space between  (Fawcett, 1958).  In  in  a layer  The  lytic  and  action  to penetrate  H a r t r e e and  i s a lysosome  Bernstein,  1969;  and  Srivastava, or  Allison  lysosome-like and  Hartree,  o f t h e a c r o s o m a l enzyme e n a b l e s t h e egg  envelopes  1974).  \  (McRorie  and  8 Because enter  attach  Hancock, at  37°C  and  h a v i n g knobbed  t o t h e zona 1965);  White,  the b u l l  The  1972).  from  is closely  The  the a n t e r i o r  appears  from  t o be  1955a; A u s t i n described murine  as  1970),  and  resistant  the  Bishop,  site  projection  Phillips  by  spermatozoa. space" instead  1966).  between  perforatorium  1963a)  and  has  spermatozoa  1966a; Hancock,  might  been  of  1966;  ungulates  appears  Conversely,  t h e r e was play  the  ( C l e r m o n t et. a_l. ,  perforatorium  that  the  characteristics  membrane o f  1966).  in  It i s distinct  The  (Hadek,  the  no  mechanical  " p e r f o r a t o r i u m " i n mammalian  T h e r e f o r e , they proposed of  the  cytological  of the d o m e s t i c  which  (Saacke  1953).  lies  i n the  semen  of s t e r i l i t y  n u c l e a r membrane  (Hancock,  of  to f e r t i l i t y  Hancock,  The  and  incubation  of the i n n e r  (1969a) argued  the term  (Buttle  cause  and  Walton,  structure  fertilization.  a b n o r m a l i t y of  1953;  1958).  while that  fibrous  implied  an  of cytoplasm  ( B i s h o p and  as a c o n e - l i k e Fawcett  related  a pyramidal s t r u c t u r e  rodents  Fawcett,  than of  and  after  to  to penetrate  edge o f the n u c l e u s .  a modification  acrosome r a t h e r  o f t h e ovum  1966a; H a n c o c k ,  a layer  impair  perforatorium  the acrosome i n o r i g i n , Walton,  the acrosome  are unable  inherited  Hancock,  Perforatorium.  originates  role  pellucida  Furthermore,  ( D o n a l d and  ( B i s h o p and  might  acrosomes  a c r o s o m e i s an  a c r o s o m e and  defect  acrosome r e t e n t i o n  post-thawing  spermatozoan  c.  d e p e n d s upon  t h e ovum, an a c r o s o m a l  Spermatozoa or  the spermatozoon  the term  " p e r f o r a t o r i u m " i n mammalian  "subacrosomal spermatozoa.  9 d.  The  sheath  Postacrosomal has  been e s t a b l i s h e d  techniques with 1961).  The  portion  of  thin  which  the p o s t a c r o s o m a l  to  be  called  Beams,  the  1935;  inexact  lateral  sheath  distance,  the  and  sheath  nuclear  ring,  located  on  extended  workers Walton,  that was  postacrosomal  Birch-Andersen,  covers  the  and  by  posterior  membrane u n d e r l a i d  postacrosomal  term  found  (Hancock,  t o be  the  Hancock  and  the e q u a t o r i a l  the acrosome w i t h  the  the  nucleus.  that  1966a) r e p o r t e d t h e p r e s e n c e  of  1966).  c o n f i n e d to  acrosome f o r a  1952;  with  (Gatenby  " p o s t n u c l e a r cap"  the base of  the  lamina  Hancock,  in his description  under  a  I t used  workers  1965;  by  dense  I t o , 1965).  several  the  except  which corresponded  the boundary  and  Birch-Andersen,  (1961) c l a i m e d  sheath  several  Bishop  the  of the  thin-sectioning  (Blom  (Fawcett  (1958) a r g u e d  Rahlmann  postacrosomal  1956;  is called  Blom and  because  of  It is a cell  a s p e c t s of the n u c l e u s  Although  the use  " p o s t n u c l e a r cap"  However, F a w c e t t was  by  presence  specialization  acrosome.  layer  The  microscopy  postacrosomal the  dense  electron  or  and  Sheath.  the  short Trevan, of  the  segment,  postacrosomal  sheath. The  chemical  obscure. than  t h e a c r o s o m e and  The  Walton,  Cell  originates  the p o s t a c r o s o m a l  seldom  as  sheath to  is  still  distortion  shows s e p a r a t i o n s f r o m  the  nucleus  1966a).  Membrane.  from  spermatozoon cell  of  However, i t i s much more r e s i s t a n t  ( B i s h o p and e.  nature  that  of  The  cell  the  spermatid.  a continuous  membrane d i f f e r s  between  membrane o f  membrane. t h e head  the  spermatozoon  I t envelops The and  the  composition the  tail  entire of  parts  the  10 with  respect  to agglutinating  properties  1966a).  The c e l l  membrane  i s composed  complex  and f o r m s  a triple  layered  Birch-Andersen, The lateral cent  cell  and a n t e r i o r  segment  acrosome,  membrane  (Blom and  the a p i c a l  of the a p i c a l  (Bedford,  owing  1963c,  part  sheath,  part  t h e membrane  i t i s anchored f i r m l y ,  to the nuclear  Pedersen, extends  1972c).  The  membrane  The n u c l e a r  f o r a variable  (Nicander  sheath  ring  and t h e main  cell  membrane  Blom  o f t h e main  seg-  structure  of the  Posterior i s closely  i s loosely  ( S a a c k e and A l m q u i s t ,  The S p e r m a t o z o a n  The anterior  neck  edges  of the  the postacrosomal  and B i r c h - A n d e r s e n , 1965;  into  attached surface  t h e neck  that  region  to the m i t o c h o n d r i a l of the Jensen's,  1964b).  o f mammalian s p e r m a t o z o a that  from a b a s a l  base o f t h e head  associated  Neck  end o f t h e t a i l  originates  to the  and B i r c h - A n d e r s e n , 1 9 6 5 ) .  and a n c h o r s a t t h e l a t e r a l  annulus  Fawcett,  e n v e l o p e forms a s c r o l l  d i s t a n c e back  and Bane, 1962a;  with  (Blom  the subja-  1969a;  to the v u l n e r a b l e  hand,  at i t s  1965b; Blom and  and B i r c h - A n d e r s e n , 1 9 6 5 ) .  on t h e o t h e r  part  I t may become l o o s e f r o m  t h e p o s t a c r o s o m a l s h e a t h , and a t t h e l a t e r a l  posterior  It  apposes  f o r a considerable  ment o f t h e a c r o s o m e (Blom  unit  lipid-protein  1965; F a w c e t t and P h i l l i p s ,  1970) and d i s a p p e a r s  4.  edges.  i n the r e s t  Birch-Andersen,  with  closely  o f t h e acrosome  membrane  of a  and W a l t o n ,  1965).  membrane  structure  (Bishop  (Bishop  i s the s h o r t  connects the t a i l  body  that  and W a l t o n ,  i s partly 1966a).  with  the head.  embedded i n t h e The s t r u c t u r e o f  11 this  region i s a very  t o z o o n and i s p o o r l y The which  complex  of  the  i s attached anteriorly s u r f a c e of  the dense o u t e r connecting  posteriorly supported  the  fibers  p i e c e has  which conforms  neck  of  the  flagellum  with  side  two  The  main c o l u m n s On  aspects  joined  five  Further into  i n the  two.  fibers  posterior,  Thus,  (Kojima,  each  1962;  Hancock,  1957a; Rahlmann,  bodies  the base of  1957;  Fawcett,  could  not  dinal  sections,  mately bands  13  1958,  verify  to  (Blom  14 and  1964b; F a w c e t t , Stefanini, verse  Although has  the  1961)  Blom and  the c o n n e c t i n g  and  Wu  and  each  of  are f u n n e l -  the  nine  (Fawcett,  1975).  the chemical  nature  two  of  the columns  to the c r o s s - s t r i a t e d  1966;  In of  longituapproxilight  Almquist,  fine  cross-banded  are b e l i e v e d  rootlets  1960)  Zamboni  b a n d s has  the  dense  (Anberg,  narrower  S a a c k e and  Newstead,  divides  granule-like  granules.  with  ventral  some w o r k e r s  Birch-Andersen,  1960;  the  and  columns.  p i e c e s a r e composed  Birch-Andersen,  and  investigators  the  The  region  main c o l u m n s  three  dense segments a l t e r i n g  1965;  that  minor  join  to  capitulum i s  Although  found  of  1970).  i t s dorsal  two  head, o t h e r  1962;  not been e s t a b l i s h e d ,  analogous  the  1966).  the o c c u r r e n c e  1971),  striations  by  a l t o g e t h e r nine columns  (Hancock, at  of  covers  posteriorly  the b a s a l p l a t e  columns.  m a i n stems a r e  piece  a convex a r t i c u l a r  the columns.  by  and  that  (Fawcett,  shaped, . t r a n s v e r s e s t r i a t e d the  sperma-  i s the c o n n e c t i n g  implantation fossa  t o the c o n c a v i t y of  either  the e n t i r e  to the b a s a l p l a t e  the c a p i t u l u m ,  connected  on  of  understood.  m a j o r component  the o u t e r  portion  to  and trans-  columns be  a s s o c i a t e d with  the  12 basal nate  bodies  of e p i t h e l i a l  contraction  neck may  cilia  and e x p a n s i o n  initiate  (Fawcett,  1975).  The  alter-  o f two l a m i n a t e d c o l u m n s  the f l a g e l l a  wave  (Saacke  i n the  and A l m q u i s t ,  1964b). Immediately centriole Fawcett, The  1955; A n b e r g ,  in  the w a l l s  in  mature  inner  spermatozoa  although  site  1958).  centriole  (Fawcett,  near  center  of the spermatozoan  envelope  mitochondria projecting  of the middle are often  nal  the i n t e r i o r  (1970) s u g g e s t e d functions. remains  that  originate 1955). cen-  and t h e  from  the mitoof  redundant  i n t h e neck r e g i o n .  small projections  between  Because  the l o n g i t u d i -  of the connecting p i e c e ,  Fawcett  t h e c o n n e c t i n g p i e c e had e n e r g y - r e q u i r i n g  The f u n c t i o n a l  obscure.  Fawcett,  the proximal  p i e c e and a s c r o l l  located  send  into  and  motility.  the m i t o c h o n d r i a columns  c e n t e r nor the  and F a w c e t t ,  that  i n the  Therefore,  t h e b a s a l body o f t h e f l a g e l l u m  A few f l a t t e n e d  nuclear  be f o u n d  (Woolley  (Burgos  (1971) s u g g e s t e d  was p o s s i b l e  sheath  and I t o ,  1975).  the k i n e t i c  the proximal c e n t r i o l e  dense  i s absent  f i l a m e n t s of the f l a g e l l u m  triole  chondrial  with  centriole  occasionally  wave  1960).  tubular t r i p l e t s set  (Fawcett,  of the f l a g e l l a  Zamboni and S t e f a n i n i  nine  1965; F a w c e t t  i s neither  The l o n g i t u d i n a l  structure  The d i s t a l  the remnants might  of the o r i g i n  1973). from  cylindrical  a s p e c t o f the n i n e columns  the d i s t a l  ( B u r g o s and  1957; Blom and B i r c h - A n d e r s e n ,  dense c e n t e r , w i t h  (Fawcett,  there i s the proximal  of the connecting piece  i s a typical  and a l e s s  1965),  the capitulum,  i n the i n t e r i o r  centriole  walls  beneath  significance  of the s c r o l l ,  however,  13 5. . The S p e r m a t o z o a n The three  tail  parts,  piece. scope  several  fine  (1948),  Schnall  a.  The A x i a l F i b e r  Bundle.  outer  nine  fibers nine  inner  fibers nine  flagella  9 + 2 The  doublets  fiber  of  by Reed and (1955)  using  bundle of arranged  pattern  ( B r a d f i e l d , 1955; A n b e r g , 1960).  The  t h i c k e r and more d e n s e  complex.  pattern  This  than  kingdoms  are double  or double microtubules;  cells  in cilia which  (Manton e_t al_. ,  The axoneme b e g i n s  t o t h e end p i e c e  fibers  are called  pattern  of a l l motile  and a n i m a l  1954).  (9+2)  b u t i t does  somewhere i n t h e e n d p i e c e  nine  concen-  a c e n t r a l p a i r and i s  and c e n t r a l p a i r  the p l a n t  and e x t e n d s  inner  around  pattern  filament  and P o r t e r ,  single microtubules Andersen,  micro-  and c e n t r a l p a i r .  i s the b a s i c  throughout  neck r e g i o n  fiber  fibers  and  Fawcett  each  are considerably  axoneme o r a x i a l  the  and M c K e n z i e  1958; Blom and B i r c h - A n d e r s e n ,  the  1952;  has b e e n c o n f i r m e d  The a x i a l  i n two rows o f n i n e  Fawcett,  occurs  and end  s t r u c t u r e c o n s i s t i n g of  s p e r m a t o z o a c o n s i s t s o f 20 f i b e r s  1957;  The  into a tuft  into  microscope.  as t h e 9 + 9 + 2  inner  piece,  by means o f t h e l i g h t  ( 1 9 5 2 ) and Wu  known  the  principal  A complex  of the t a i l  electron  i s differentiated  occasionally frayed  fibrils.  the  trically  piece,  (1888) o b s e r v e d  filaments  mammalian  spermatozoa  i . e . the middle  the t a i l  exceedingly  Reed  o f mammalian  Ballowitz that  Tail  (Anberg,  t u b e s known  i n the not r e t a i n 1957).  as the  t h e c e n t r a l p a i r c o n s i s t s o f two  ( B r a d f i e l d , 1955 ; A f z e l i u s , 1959; Blom and B i r c h -  1960; T e l k k a  e t a l . , 1961; H a n c o c k ,  1966).  The  14 doublets  consist  microtubule  and  section  with  Subunit  A  A has wall  two  arm  the  links  1970)  and the  Fronk, 13  still  Telkka  spoke pair  i s made up  that  13  B i s composed  joins  (Gibbons,  with  adenosine  1965;  Although the  The  dynein  The  (Stephens, sheath Rahl-  wall  of  the  central  the  of  (ATPase) Gibbons  pair  sheath  flagellar  seems t o c o n v e r t  1970;  arms c o n s i s t  Rowe, 1965;  in a helical  the mechanism o f  of  slender  t u b u l i n s (Warner,  1974).  G i b b o n s and  i s enclosed  two  1959;  cylindrical  triphosphatase  of  end  doublets  Afzelius,  outer-  protein protofilaments,  10  Klug,  Each m i c r o t u b u l e  t u b u l i n s and  obscure,  Amos and  The  i t to a h e l i c a l  1957;  about  from i t s  1959;  the  s u b u n i t A has  tubulins, of  t i p , subunit  (Afzelius,  adjacent  struc-  clockwise  at  B  1961).  e_t a l . , 1 9 6 1 ) .  Each  (Anberg,  of  the  A.  subunit  a hollow  appendage p r o j e c t i n g  i t to the  subunit  than  T e l k ka e t a l . ,  i n t h e row  T e l k k a e t a l . , 1961).  1972).  1972c).  1960;  that connect  a protein  activity  doublet  complete  the  tail  l o o k i n g toward  i n n e r arm.  e t a l _ . , 1973;  dynein,  of  the  central  subunit  Tilney  next  a radical  mann, 1961;  and  the  r a t h e r than  1960;  base  a flag-like  towards  subunit A  Grimstone,  Grimstone,  has  nexin  around  c e n t e r of  a  in cross  w a l l of  s h o r t d i v e r g i n g arms t h a t p r o j e c t  G i b b o n s and  the  i s C-shaped  to the  i t is solid  flagella  a s u b u n i t A,  B which  to the  and  and  the  toward  most arm  a subunit  1961)  from  subunits:  i t s ends a t t a c h e d  (Gibbons  Viewed  two  i s closer  (Rahlmann, ture  of  and  i s composed (Pedersen,  movement i s  the c h e m i c a l  energy  o f a d e n o s i n e t r i p h o s p h a t e (ATP) i n t o m e c h a n i c a l e n e r g y and i n d u c e t h e s l i d i n g of neighboring doublets ( S a t i r ,  1965;  Gibbons,  1965,  1975 ;  Summers and G i b b o n s , 1 9 7 1 ) . I t has b e e n r e p o r t e d t h a t i m m o t i l e s p e r m a t o z o a  15 have no dyne i n Pedersen  arm and c a u s e  and Rebbe,  Peripheral are  with  fibers  1961).  (Saacke  proposed triole,  that  area  part  (Blom  of the p r i n c i p a l  and B i r c h - A n d e r s e n , each  fibers  1964b).  (Blom  differ  and s h a p e .  from  from  differences  initial  and  (Fawcett, The toward  one a n o t h e r  (1955),  1960).  doublet.  i n the cross  fiber  number  1,5,  are d i s t i n c t l y  larger  9,1,5, and 6 a r e  There  are also  marked  configuration,  e x t e n t o f the dense  dense  fibers  a r e round  fibers  at the outer contour  the corresponding doublets medulla  and a t h i n  on t h e s u r f a c e r e p l i c a s  suggest  that the  o f each  i n the c r o s s s e c t i o n a l  longitudinal  (1958)  1970).  have a t h i c k tions  dense  Fawcett  later  the wall  I n many s p e c i e s ,  and B i r c h - A n d e r s e n ,  interspecific size  1960;  the d i s t a l cen-  (1969b) s u g g e s t e d  as o u t g r o w t h s  fibers  from  t h e o t h e r s , b u t i n a few s p e c i e s number  larger  there  piece  of the nine  Although  had o r i g i n a t e d  6, as d e s i g n a t e d by B r a d f i e l d  than  spermatozoa,  on t h e same r a d i u s a s t h e c o r r e s p o n d i n g  and P h i l l i p s  arose  e_t a_l. , 1975;  but these are present only i n  In c r o s s s e c t i o n s ,  these  nine dense  sectional  The  fibers  and A l m q u i s t ,  Fawcett  dense f i b e r s  and  dense  p i e c e and t h e m a j o r  are aligned  doublet  The  t o t h e axoneme o f mammalian  d e c r e a s i n g dimension  Rahlmann,  (Afzelius  1975).  nine p e t a l - l i k e  the middle  sterility  globular  medulla  also  (Fawcett,  cortical  1970).  layer.  and t a p e r The  fibers  Oblique  stria-  of the c o r t e x of the dense  fiber  s u b u n i t c o m p o s i t i o n s ( P h i l l i p s and O l s o n , has a v e r y  Although  several  cysteine  and t h e a b s e n c e  fine  investigators  periodicity  (Pedersen,  1975 ) .  1972c).  reported a high content of  o f ATPase a c t i v i t y  i n both  the medulla  16 and  c o r t e x of  Pihlaja  and  Roth,  cetti etal. cortex.  the dense 1973;  t u b u l e s was  Burton,  1973).  also  fibers  through  they might  The  be  surrounds sheath, the  the the  (Wu  and  Jensen  1949;  axial  and  of  fiber  which doubled  upon  near  that  the  1973b;  The  of the  between  flagellum  They  arise  Phillips,  elements  of the  Their  middle  1969b),  and 1970).  piece i s charac-  Because of than  from  motor  forms  which  the m i t o c h o n d r i a l  the o t h e r p o r t i o n  was  sheath  and  of  traced i t s (Hodge,  have d e s c r i b e d a d o u b l e  to form  the head.  the  Several investigators  Birch-Andersen  some s t r a n d s f a i l e d  be  1955).  1966)  the h e l i x  numbers  among s p e c i e s ( F a w c e t t ,  observed  itself  and  1970).  piece i s thicker  Newstead, and  suggested  ejt _ a l . ,  I t o , 1965 ).  bundle.  to the m i t o c h o n d r i a .  terminating  around  a m i t o c h o n d r i a l sheath  McKenzie,  However, Blom  and  different  third  (Fawcett  Phillips,  (1887) f i r s t  VJu and  and  fibers  presence  the middle  tail  origin  the a n t e r i o r  accessory tensile  (Fawcett  by  (Baccetti  present i n limited  M i t o c h o n d r i a l Sheath.  terized  (1958) and B a c -  motility.  Fawcett  the dense  numbers a r e m a r k e d l y  b.  Nelson  1973a;  t h e d o u b l e t s and  detected  are  ( T e l k k a et. a_l. , 1961; the c o r t e x of  between  spermatozoan  fibers  apparatus  1973).  e_t a_l. ,  T h e r e f o r e , a c o n t r a c t i o n of dense f i b e r s might  i n the  Satellite  and  Price,  activity  central  the dense  (Baccetti,  (1975 ), however , d e m o n s t r a t e d ATPase a c t i v i t y i n t h e  ATPase  involved  fibers  (1960) r e p o r t e d a s i n g l e a loop with  S a a c k e and composed  to reach  helix.  i t s two  Almquist o f one  t h e end  free  (1964b)  o r more  chain ends  also  strands  of the middle  piece.  17 In g e n e r a l , and  a r e a r r a n g e d end t o e n d i n a s i n g l e  flagellum  (Anberg,  The duality form and  the mitochondria of the middle  1957; F a w c e t t ,  helix  but retain  of the elements  The l e n g t h  of the middle  t o t h e number o f m i t o c h o n d r i a l  different  among s p e c i e s  organization  the expanded  space  1970).  spermatid  differences  among s p e c i e s  ( p s e u d o m a t r i x ) and  (true matrix).  species-specific  the f u n c t i o n a l i s obscure,  energy  However, b u t show  requirement  surrounds  spermatozoa  of spermatozoa  (Jensen's r i n g ,  ring  centriole).  bundle  piece  and p r i n c i p a l  piece  o f mammalian s p e r m a t o z o a  1887;  Blom and B i r c h - A n d e r s e n , 1 9 6 0 ) . packed  at the junction  The  fiber  form  of those  be r e l a t e d t o  the a x i a l  The  normal  implication  i t might  some  to  fertilization.  The A n n u l u s  closely  mitochon-  tend t o s e p a r a t e to  i n the mitochondria sheath of Although  i s pro-  extensive internal r e -  do n o t show c o n s p i c u o u s p s e u d o m a t r i x  (Fawcett,  achieve  matrix  which  During spermiogenesis,  cristae  space  i n the  gyres, i s remarkably  In the e a r l y  intracristal  intercristal  intracristal  c.  1962).  t h e two membranes o f f o l i a t e  condensed  the  (Andre,  indivi-  variations  piece,  (Fawcett, 1970).  the m i t o c h o n d r i a o f s p e r m a t i d s undergo  species  their  ( F a w c e t t , 1962; F a w c e t t  portional  form  around the  ( F a w c e t t , 1958) and t h e r e a r e s p e c i e s  I t o , 1965).  are elongate  1958, 1965, 1 9 7 5 ) .  mitochondria are not fused  and a r r a n g e m e n t  dria,  piece  among t h e s p e c i e s .  I t might  sections  of the middle (Jensen,  I t i s composed o f  filamentous subunits oriented  of the l o n g i t u d i n a l  annulus  circumferentially.  of the annulus  be t r i a n g u l a r  i n some  varies  species  with  the base  projecting  inward toward  mitochondrial species  a d h e r i n g to the c e l l  helix  i t might  be  and  t h e dense fibrous  might  caudal The  traverse  end  shed  annulus appears during  to  The C y t o p l a s m i c D r o p l e t .  described middle  first  piece  droplet  by R e t z i u s  ground  c y t o p l a s m , a l l bounded  1964),  1921;  1961).  (Harrison  Glover,  sloughed  and  (Fawcett,  internal  some l a r g e r  some l i p i d ,  (Dott  and  and  of the  Bloom  fine,  and  a  (Gatenby  and  c a r b o h y d r a t e (Mann,  Dingle,  1974)  membrane  1955;  and  material:  vesicles,  by a d i s t i n c t  the  1970).  structure  of the G o l g i  and F a w c e t t ,  and W h i t e ,  the r o l e  1968),  glycolytic  phosphatases  of the c y t o p l a s m i c  droplet  n o r m a l l y moves down t h e m i d d l e p i e c e  o f f during  epididymis,  displacement of  (Moniem  1972).  Although droplet  The  It contains  l y s o s o m a l enzymes  enzymes  the  Burgos  1964b).  t h e neck  tubules,  and Woodger,  at the  ( 1 9 0 9 ) , b o r d e r s on  of d e r i v a t i v e s many s m a l l  droplet  droplet,  of spermatozoa.  consists  Nicander,  The  cell  interruption.  cytoplasmic  curved  and  movement  other  The  ( S a a c k e and A l m q u i s t ,  prevent caudal  w i t h the  In  (Fawcett, 1970).  the c y t o p l a s m i c  the t a i l  apex  i n contact  to the annulus without  of the middle p i e c e  mitochondria  d.  and  fibers  the  sheath l a t e r a l l y .  semicircular  membrane a d h e r e s c l o s e l y It  membrane and  though  i s obscure, and i s  the passage of  spermatozoa  through the  some e j a c u l a t e d  spermatozoa  retain i t .  There are c o n f l i c t i n g  reports  the  o c c u r r e n c e o f the d r o p l e t  the  p r e s e n c e and  location  about and  the r e l a t i o n s h i p  between  the degree of m a t u r i t y ,  of the d r o p l e t  a r e t a k e n as  an  but  19 approximate  index  of spermatozoan m a t u r i t y .  hand, White  and Wales  (1961) r e p o r t e d t h a t  epididymal  spermatozoa  attachment  of the c y t o p l a s m i c  e.  to cold  The F i b r o u s S h e a t h .  spermatozoa contains  two l o n g i t u d i n a l  p i e c e o f mammalian  by t h e f i b r o u s  sheath.  elements  travel  which  of the p r i n c i p a l  1962,  1965; T e l k k a e t a l . , 1961; S a a c k e  of  1970b).  closely  ribs  that  spaced  piece  the a x i a l  The  sheath  flagellum The  attachment  caudal  (Fawcett  and P h i l l i p s ,  elastic  1970).  p r o p e r t i e s which  resisting  the s l i d i n g  (Fawcett,  1970,  f  *  ribs  induce  t h e axoneme.  1964b). columns piece  from  to f i b e r s  might  t h e axoneme longitudinal  possibly flagellar  propagated  closer  the t i p of the  and v e n t r a l  (terminal piece).  the t e r m i n a t i o n of the f i b r o u s  axoneme i s b o u n d  from  along  The c o n t i n u o u s  movement  columns  along  endow  the sheath  bending  by  t h e axoneme  1975).  The End P i e c e  beyond  semi-circular  becomes t h i n n e r and  of the sheath  c o l u m n s and i n t e r s p a c e d p e r i o d i c with  series  p a r t of the p r i n c i p a l  displacement  1958,  i s made up o f a  and A l m q u i s t ,  of the dorsal  both  1964b;  end t o two l o n g i t u d i n a l  (Saacke  on  and A l m q u i s t ,  ends a b r u p t l y 2 t o 3  i n the b u l l  down  sheath  1955; F a w c e t t ,  f i l a m e n t as i t moves p o s t e r i o r l y  then  The  interconnected  The s h e a t h  3 and 8 i n t h e p r o x i m a l prevent  sheath  and o c c a s i o n a l l y  ( T e l k k a e_t aJ,. , 1 9 6 1 ) . to  (Bradfield,  The f i b r o u s  attach at e i t h e r  with the  droplet.  sides  Pedersen,  the r e s i s t a n c e of  shock was c o r r e l a t e d  The p r i n c i p a l  i s surrounded  On t h e o t h e r  The e n d p i e c e i s e x t e n d e d sheath;  o n l y by t h e c e l l membrane.  piece i s q u i t e v a r i a b l e depending  t h e r e f o r e , the  The l e n g t h o f t h e end  on t h e s p e c i e s ( F a w c e t t ,  1970 ) .  20 C.  MATERIALS AND Standard  age  d a r k male mink  were u s e d  During  the  METHODS  f o r the  barbital the  from  anaesthesia.  cauda e p i d i d y m i s  26  Small and  immediately  solution  were r e l e a s e d by  squeezing  t o z o a were c o n c e n t r a t e d  (Jones, the  by  s o d i u m c a c o d y l a t e ) , washed and  rev/ashed  concentrated  (1500G,  and  i n Epon 812  embedded  glass  knives  uranyl  acetate  Coggeshall, mission  using  an  i n 1%  1965)  electron  and  15  1975,  of  under  1971)  mM  times,  (Jones,  1971).  supported  (Luft,  1961).  acid  and  examined with  microscope.  sliced  i n 1 ml  of  suspended  (700G,  from Krebs-  spermatozoa  15  The  sperma-  min), and  fixed  150  i n 3%  s a m p l e s were agar,  mM  double  again  dehydrated,  S e c t i o n s were c u t  with  s t a i n e d by  lead c i t r a t e an  pento-  p o s t f i x e d i n osmium  ultramicrotome,  acetic  sodium  glutaraldehyde  three  of  reproductive  where t h e The  years  spermatozoa.  t i s s u e were  immersed  tissue.  (100  min),  LKB  19,  centrifugation  glutaraldehyde-cacodylate  tetraoxide,  of  male mink pieces  three  study  to March  five  Henseleit-Ringer  in  more t h a n  ultrastructural  period February  t r a c t s were r e m o v e d  of not  AEI-Corinth  (Venable 275  5% and  trans-  21 D.  RESULTS  1.  The  Spermatozoan  The ovate  h e a d was  Head  found  in outline,  with  t o be an  dorsoventrally flattened  average  length of  5.83  and  .urn and a b r e a d t h  o f 0.35 um i n l a t e r a l v i e w a t t h e e q u a t o r i a l segment ( F i g s .1,2 Table  3).  In  sagittal  major p a r t of thicker  than  consisted  the the  of  an  s e c t i o n s , where  head,  the  anterior electron 20  nm  posterior  part  the  p a r t of  (Fig. 1).  approximately  diameter  in  s i z e , were s c a t t e r e d t h r o u g h o u t i n the  posterior  portion (Fig. 1).  thirds  of  head,  nucleus  containing  a less  the  dense  posterior  o n e - t h i r d of  acrosomal  sheath.  nucleus,  the  segment,  0.68  a  beyond  little  1.61  jam  long,  segment, and  matrix, tions  The  3,4).  (acrosomal  from  the  covered  At  the  largest  nucleus;  apical  the  0.34  jam  the  proximal  s e c t i o n s , the  s w e l l i n g , F i g . 1)  1,3).  The  the  volume  extended  segment,  the  equatorial  (Figs.  1,39  amorphous  the  a bilateral a connected  sec-  equatorial  acrosome appeared  as  the  apical  and  o f an  post-  around  the  area  of  two-  acrosome  with  and  increased  in longitudinal  border  producing formed  an  the  s m a l l e s t area  long  vacuoles  anterior  t h e main  segment;  and  nucleus,  with  i n three parts:  p e r f o r a t o r i u m , composed  in sagittal  separated  the  the  long, occupied  triangular  (Figs.  segment,  t i p of  with  (Figs.  upon a c r o s o m a l  long, occupied the  jam  3).  was  head was  Depending  followed  1.55  Table  the  was  vacuoles, varying  In t h e  covered  amorphous m a t r i x  acrosome appeared jum  was  nucleus  a  nuclear material  The  nucleus  number  the  occupied  a number o f  (Fig. 1). the  the  The  d e n s e mass w i t h  of  nucleus  and  to  be  swelling hump-like  22 structure  in frontal  posterior  t o the e q u a t o r i a l  also are  formed  a bilateral  present  was  ( F i g . 1).  (Fig. 2).  segment,  swelling.  i n the head.  amorphous m a t e r i a l swellings  sections  Immediately  the  A  postacrosomal  total  of  six swellings  Some d i s i n t e g r a t i n g  observed  Although  i n t h e empty  membranous space  t h e a c r o s o m e were d i s l o d g e d , t h e  torium,  swelling,  remained 1.99  intact  nm  l o n g and  identifiable and  Table  connecting The nucleus  on  3).  implantation  both  (Figs. a 6 nm  layer  the o t h e r p a r t  The  caudal  fossa  piece  the c e l l  s u r f a c e of  t o accommodate  (Figs.  that  i t was  i n n e r and  distinguishable appeared  t o be  The  outer  acrosomal  membranes  separated  from  except  f o r the  sheath  (Fig. 1). the c e l l  membrane f o r m i n g  At  was  membrane  (Fig. 1  formed  the  the  so c l o s e l y  opposed  (Fig. 3).  to  the  However,  of  the  n u c l e u s where  they  ( F i g . 5).  b o u n d e d by  distinct  The  cell  portion  and  the d i s t a l  the p o s t e r i o r membranes  the  end  o u t e r and  membrane was  i n the acrosomal  membrane was  membranes were u n i t  head  was  nucleus  to i d e n t i f y  (Fig. 3).  apical  the  n u c l e a r membranes became  apart  the c e l l  perfora-  the c a p i t u l u m of  a t t h e c a u d a l end  a c r o s o m e was  the  and  5,6,7).  difficult  4 nm  the  sheath  of dense m a t e r i a l  the i n n e r a s p e c t of The  of  postacrosomal  i n n e r n u c l e a r membrane was  the  sheath,  and  1,4).  of  or  the c o n t e n t s of t h e ' a p i c a l  t h e main p a r t s o f acrosomal  sheath  part  of  the  inner usually head  postacrosomal  of  anchored  the  postacrosomal  firmly  ring  ( F i g . 7).  (Figs.  1,3,4).  on  the  nuclear  A l l spermatozoan  23 2.  The S p e r m a t o z o a n The  s p e r m a t o z o a n neck  striated  and s c r o l l s  base o f the head, posteriorly  posteriorly  immediately striated  with  it  appeared that plates  The c r o s s  of  and t a p e r e d  nm  structure, lay  and t h e c r o s s was  but separated  consisted  The c r o s s  light  continuous  on b o t h  striated  column (Fig.  5,6).  (Figs.  parallel of cross aspect  appeared d i s c o n t i n u e d  bands  striations  t o the  Because  t o each other,  of  5).  Individual f i b e r s of  distally  the c i r c u l a r  was  o f 43 nm t h i c k d e n s e bands.  sides  o f a d o r s a l and  end, a d j a c e n t  form a "connected r i n g " addition,  associated  a t the proximal  were a l s o a r r a n g e d  fibers  an a r t i c u l a r  were composed 6.6  i n the  p l a t e was  and c o n s i s t e d o f 10 t o 13 s t r i a t i o n s  striations  At the  supported  plate confined  Because the c a p i t u l u m  5,6).  a few  5,7,8).  membrane was  p l a t e above  the capitulum  c o l u m n were w i d e s t  fibers  (Figs.  cross  by means o f a d i f f u s e d e l e c t r o n  and v e n t r a l a s p e c t s  a l t e r n a t e with  capitulum,  In  the b a s a l  (Figs.  centriole,  The b a s a l  the capitulum  between  the d o r s a l  to  (Fig.5).  columns below.  long  nuclear  The c a p i t u l u m ,  in  the  the outer  fossa  dense m a t e r i a l .  which  o f the capitulum,  o f membrane  by a 48 nm t h i c k b a s a l  implantation  0.7 ^m  consisted  l o n g i t u d i n a l columns, proximal  mitochondria,  ventral  Neck  they  these appeared  ( F i g s . 5,9).  the connected  ring  by a d o r s a l , a v e n t r a l and two  l a t e r a l v e r t i c a l c l e f t s ( F i g s . 5,6).  Thus, f o u r c r o s s s t r i a t e d  c o l u m n s were f o l l o w e d f u r t h e r d i s t a l l y i n t h e neck by two m a j o r and f i v e m i n o r c o l u m n s ( F i g s . 5 , 6 , 9 , 1 1 ) . Below t h e neck r e g i o n , i t a p p e a r e d  24 that  each major column  five  minor  columns a t o t a l  n i n e dense  fibers  Immediately the  proximal ring  triole  lay at  in  triplets  nine  thus  forming  with  columns connected  i n n e r s u r f a c e of  situated The  to  i n the  longitudinal  t h e main a x i s  were o b l i q u e l y  the c e n t r i o l e  central  found  i n the  (Fig.  11).  tions  revealed  axoneme  the  to  giving  the  the  capitulum,  center  of  a x i s of  the  the  (Fig. 5).  inserted  con-  cen-  The  at a r e g u l a r  a whirl-like  two  the  anterior  Enclosed  by  12,13).  In  margin of the the  cell  nine  spacing  appearance i n  by  A  and  formed  of  neck  the  the  by  the  microtubules  of  membrane on  the  helix  s i d e s of the  appeared  as  usually  which  (Figs.  secthe  neck r e g i o n , t h e r e was  mitochondria  also  columns  region, cross  oriented mitochondria  marked  7,10).  neck,  scrolls  there  (Figs.  7,8).  Tail  of  of  the  a cylindrical  the m i d d l e  axial  c o n s i s t e d of  microtubules.  t h e axoneme was  bundle  membranes w h i c h  pair  of  of double  w h i c h v/ere c o n n e c t e d  surrounded  subfiber  level  presence  arrangement  central  diameter  the  transverse section  9 + 9 + 2  double  the  Spermatozoan  The  of  longitudinally  were r e d u n d a n t The  of microtubules  a lower  (Figs.  or  pair  interior At  one  The  was  70°  two,  sections ( F i g . 7). The  3.  of  the  5,7).  about  into  5,12,13).  beneath  (Figs.  the w a l l of  cross  (Figs.  centriole  nected  tubular  split  two by  Each double  f i l a m e n t bundle single  the  bundle  p i e c e showed  of  nine  their  sheath  uniformly  microtubule  B s h a r i n g a s e c t o r of  ( F i g . 14).  microtubules  central  a  of  28  nm  and spaced  c o n s i s t e d of  wall  (Figs.  14-17).  25 Subfiber with  A was  nearly  a complete  circular  homogeneous d e n s i t y .  w h i c h were d i r e c t e d t o w a r d doublet.  The  a distinct m e t e r and A was  (Figs.  appeared  complex In the  found  (Fig.  while  the  inner  14).  The  shape  the  by  were  length  3 and the  inward  the  Small  the  between  the  fibers  the  B was  the  outer  of  of  27  inward nm  in  Subfiber Each  centrally  spokes,  electron axial  by  dia-  axoneme.  these  the  arms  neighbouring  spoke d i r e c t e d  Slight  piece  appeared  appeared of  the  fibers the  there  dense filament  smooth  dense  piece  rest  ( F i g . 15).  of  polygonal  dense f i b e r s  were  of  the  the  the  distally, doublets  principal  satellite and  and  around  piece  The  Dense  were  each  of  at d i f f e r e n t (Figs.  inner  dense  15,  border  along fibers  replaced  dense  f i b r i l s were a l s o the  dia-  diminished  l o n g i t u d i n a l elements  Further  (Fig.  different:  forms.  14-16).  fibers  sections,  larger in  gradually  to  The  irregular  fibers  (Figs.  to corresponding part  or  w h i c h were b o n n e t  principal  of  in cross  f o r m s and  then  peripheral  dense f i b e r s .  serrated  outset,  the  regions,  were 9 o u t e r  6 were e l l i p t i c a l  extension  close  or  there  size  before  terminal round  and  other  sheath  terminated  the  and  projected  of  of each  principal  border  l a r g e at of  a radial  ( F i g . 14).  diameter)  micrographs.  center  midpoint  dense f i b e r  and  the  8 ended  fibrous  of  of  9, 1, 5,  fibers  the  with  B of  arm  i n the  nm  inner  Subfiber  hollow  between  (9 + 2 ) ,  border  than  outer  (29  14).  outer  meter  the  m i d d l e and  axoneme  number  At  subfiber  the  toward  thickening  was  the  t o be  connected  14-17).  a slight  of  I t had  ( F i g . 14).  tilted  A was  material  end  appendage  slightly  subfiber  was  distal  the  tubule  the fiber levels 16).  found of  the  26 fibers  ( F i g . 14).  In  the  surrounded sheath  middle by  a single  (Figs.  with  about  dria  varied  p i e c e r e g i o n , the  10-14,  53-57  sections, to  ( F i g . 21).  coarse on  the d i s t a l  (Fig.  as  a triangle  of  p i e c e was  some  the  Cytoplasmic  middle  piece  composed  Mitochon-  of  In  non-  longitudinal  the base  juxtaposed the  d r o p l e t s were  i n some  long  appeared  ( F i g . 23). with  10 jum  ( F i g . 10).  and was  mitochondria  p r o j e c t e d downward t o f a c e  i t s apex.  end  annulus  coiled  were  outer  observed  spermatozoa  23). In  the  surrounded  (Figs.  principal by  ferentially  two  at  10, 21, 22)  The  membrane.and  fibers  middle  dense m a t e r i a l  i t appeared  the c e l l  The  of mitochondria  (Figs.  homogeneous e l e c t r o n  of h e l i c a l l y  21, 2 2 ) .  gyres  in size  pseudomatrix  layer  dense f i b e r s  a fibrous  The  longitudinal  ended  of  sheath  oriented ribs  19-21).  aspects  p i e c e , the  the  were  elements  piece  ( F i g . 20)  piece  the  and In  pattern (Figs.  the of 18,  end  end the  and  marked  was of  around  their  circum-  the  ends  to  t h e d o r s a l and  15-17).  jam f r o m junction  The  the of  form ventral  sheath  t i p of the  columns  the  principal  piece.  p i e c e , an f i b e r s ) was  20).•  the  at  along  (Figs. 1.5  composed  halfway  joined  that ran  a b r u p t l y at a d i s t a n c e of  flagellum  w h i c h was  extending  ribs  principal  axonemal complex  axial  f i l a m e n t complex  surrounded  o n l y by  the  (9+2 cell  membrane  27 Figures  1 t o 4.  U l t r a s t r u c t u r e of the epididymal  spermatozoan  head. Figure  1.  Sagittal  swellings segment  sections  on t h e p r o x i m a l  (acrosomal  acrosomal  sheath  materials  (MM)  inside left,  intact  the  Figure  2.  though  The c e l l  membrane  acrosomal  head  with  (N)  (CM) i s  contains  separated  postacrosomal  connected  (AM).  (PS)  o f head  The  acrosome  surrounded membrane  membrane  by (CM),  (NM) a r e  80,000X.  s e c t i o n of the a p i c a l part  perforatorium  (PM).  (P).  The c e l l  and n u c l e a r  and a v a c u o l e  membrane  swelling  18,000X.  amorphous c o n t e n t  membranes.  showing  hump-like  s e c t i o n of the a p i c a l part  membrane,  Sagittal  matrix  of the acro-  (AE) and  triangular perforatorium  filled  4.  (P)  (AC), but i t coheres to  spermatozoon.  a c r o s o m a l membrane  Figure  and p e r f o r a t o r i u m  (AS) and t h e p o s t a c r o s o m a l  Sagittal  double  are observed  The n u c l e u s  s e c t i o n showing  a testicular 3.  membranous  At t h e lower  the content  damaged.  o f t h e acrosome  Frontal  showing is  swellings.  post-  30,000X.  swellings of  Disintegrating  swellings  bilateral  of the e q u a t o r i a l  AS) and on t h e  a p i c a l end o f t h e acrosome  sheath. Figure  even  (V).  part  showing  and amorphous m a t e r i a l s  (AO) a p p e a r s  vacuoles from  (PS).  the postacrosomal  remain  border  swelling,  the acrosomal  some  o f t h e head  containing  (V), a l l enclosed  120,000X.  of the  nonhomogeneous by t h e c l e a r  29 Figures  5 t o 8.  Figure  5.  Ultrastructure Frontal  section  of the spermatozoan  neck.  o f t h e neck r e g i o n .  The  plate  (BAP) i s c o n f i n e d  (area  between  anteriorly  electron consist  two a r r o w s ) .  with  posteriorly  to the implantation  o f dark  membrane  the capitulum  material.  the dense  (C) by d i f f u s e d  fibers  with  the  capitulum  (C) i s c o n t i n u o u s ,  the  connected  ring  (CR).  located  i n the center  nuclear  membrane  Figure  6.  nucleus. Lateral  capitulum striated Figure  7.  triplets  Inserted The  section  white  posterior  tudinally  showing  that  divides  centriole is ring.  at the caudal  a cleft  the connected  (SC).  Sagittal section  nine  but a c l e f t  The p r o x i m a l  (NM) i s s e e n  Note  A unit end o f  125,000X.  columns  sectioned  (DF) d i s t a l l y .  of the connected  (C) and a l s o  (PC)  (SC)  s t r i a t i o n s and a r e c o n n e c -  ted  the  (NM) and  The s t r i a t e d c o l u m n s  and l i g h t  fossa  I t i s associated  the nuclear  with  basal  d i v i d i n g the r i n g o f the  180,000X. showing  transversely  a proximal  and a s c r o l l  (T) a r e r e g u l a r l y  arranged  centriole (S). as a  l i n e s show t h e o r i e n t a t i o n ring  oriented  is distinctive mitochondrion  (arrow).  of  The while.  triplets. A longi-  (M) i s s e e n .  50,000X. Figure  8.  Oblique  bilaterally.  section 37,500X.  showing  scrolls  (S) s i t u a t e d  30  31 gures in  9  to  13.  Ultrastructure  of  the  neck  and  middle  piece  section  of  the  neck  r e g i o n showing  (CR)  striated  spermatozoa.  Figure  9.  Sagittal  connected Figure  10.  Axial  showing Note  ring  sections  that  the  shape to  The  helix.  12,0O0X.  11.  and  two  a central the  of  columns.  Figure  12.  axial (CO)  pair  center.  gyres  covers  an  of  of  the  the  (N)  p i e c e (IIP)  has  of  five  the  minor  filament  (M)  the  middle  no m i t o c h o n d r i a l  level  and  axial  (M).  mitochondria  surface of  upper  Mitochondria  middle  i s different; a helix  (PP)  (MC)  80,000X.  of mitochondria  the  piece  major  section  showing filament  which  entire  size  at  columns.  neck columns  complex  surround  the  (CP)  bundle  50,000X.  Cross  region  the  nucleus  section  showing  in  the  principal  Cross  region  and  (HH)  mitochondria  piece.  Figure  of  a p p r o x i m a t e l y 53  (arrow) c l o s e of  of  the  two  at a  lower  double  complex  of  microtubules  inside  i s surrounded  level  by  the  the  (DM)  bundle  mitochondria  neck of  of  the  columns  (M).  80,000X. Figure  13.  Oblique  section  region  showing  a bundle  double  microtubule  at  (DM).  of  a  lower nine  level  columns  50,000X.  of  the  (CO)  neck  and  a  33 Figures  14 t o 2 3 .  Figure  14.  U l t r a s t r u c t u r e o f the spermatozoan Cross  s e c t i o n of the middle piece  components o f t h e a x i a l fibers, rest. fiber  number Subfiber  seen.  between  and Figure of  A  (a) a p p e a r s  cell  15.  centrally.  t o be l a r g e r t h a n s u b -  arms shows an a p p e n d a g e ( a p ) Many s a t e l l i t e  o f t h e axoneme.  enclosed membrane  Cross  (CM).  16.  s e c t i o n of the p r i n c i p a l  number  Cross  piece.  sheath.  Figure  Cross piece  18.  Cross  fibrous Figure  19.  cipal  piece  Note t h a t  ( F S ) . 60,000X.  showing  37,500X.  near  the  number  9,  corresponding  75,000X.  no d e n s e  fibers.  of the p r i n 60,000X.  s e c t i o n o f t h e end p i e c e  showing  no  60,000X.  Longitudinal  structure  present  s e c t i o n of the lower p a r t  sheath.  piece  devoid  t h e dense  The r e m n a n t s o f t h e d e n s e f i b e r s ,  17.  (MH)  s e c t i o n of the middle of the p r i n c i p a l  double microtubules.  cipal  helix  3 and 8, a r e r e p l a c e d .by l o n g i t u d i n a l  1, 5 and 6, a r e s t i l l  Figure  The b u n d l e o f  80,000X.  columns of the f i b r o u s sheath Figure  f i b r i l s (sf)  by t h e m i t o c h o n d r i a l  the mitochondrial  fibers,  The d e n s e  The e l e c t r o n d e n s e m a t r i x i s a l s o s c a t t e r e d  the f i b e r s  fibers.is  bundle.  showing  9, 1, 5 and 6, a r e l a r g e r t h a n t h e  B ( b ) . The o u t e r  projecting are  fiber  tail.  s e c t i o n through  showing m i c r o t u b u l e s  of the f i b r o u s sheath  a broken  (MT).  prin-  The h e x i a l  (FS) i s o b v i o u s .  34 Figure  20.  Longitudinal  (area  between  (FS). Figure  principal  axial  hexial Figure  22.  found  filament  the  the  no  end  piece  fibrous  sheath  membrane  on  In  principal  the  (FS).  anchors  cytoplasmic  the  distal  end  of  at  the  the  principal at  droplet of  (MP)  are (left),  by  the  50,000X. mitochondria  mitochondria  section the  piece  i s surrounded  section  and  (CM)  the- m i d d l e  Some m i t o c h o n d r i a  sheath  (MP)  through  (PP).  arrangement of  piece  The  section  complex  Longitudinal  middle  (AN).  (PX).  Longitudinal  23.  cell  piece  fibrous  showing Figure  arrows) showing  Longitudinal  pseudomatrix the  through  37,500X.  21.  and  two  section  the  (M).  junction piece  a triangular (CD)  is  helix  50,000X. of  the  (PP).  The  annulus  sometimes  middle p i e c e .  37,500X.  36 E.  DISCUSSION  1.  The s p e r m a t o z o a n  Except throughout geneous. of  Head  f o r small  Irregular  the nucleus,  i n mink  of vacuoles,  condensation  The  acrosome  its  apical  i n t h e number,  the  spermatozoa  tendency  1966a).  s p e r m a t o z o a was r e l a t i v e l y beyond  f r o m man, monkey, b u l l ,  hand,  t h e n u c l e u s as i n  boar, r a b b i t ,  hare,  and Bane, 1966; F a w c e t t ,  t h e spermatozoa from  squirrel  s m a l l and  have  much  chinchilla,  larger  acrosomes  1970). elongated  cone  shape  t o remain i n t a c t  had d i s a p p e a r e d serve  than that i n  o f mink  g u i n e a p i g , and g r o u n d  The  The s i z e  and W a l t o n ,  little  (Saacke  i n chromatin  (Bishop  On t h e o t h e r  (Fawcett,  and o r i e n -  as a r t i f a c t s  of accidents  d o g , h o r s e , and c a t ( N i c a n d e r  1970).  shape,  s p e r m a t o z o a was s m a l l e r  segment e x t e n d e d  portion  f o r the b u l l  s p e r m i o g e n e s i s (Fawcett, 1958).  i n mink  homo-  and B i r c h - A n d e r s e n , 1 9 6 5 ) .  t h e y were c o n s i d e r e d  during  human s p e r m a t o z o a  may  Blom  1964a) o r r e s u l t s  the vacuole  ram,  1964a;  of the i r r e g u l a r i t i e s  and A l m q u i s t ,  of  scattered  the chromatin appeared n e a r l y  s p e r m a t o z o a as r e p o r t e d  ( S a a c k e and A l m q u i s t ,  tation  w h i c h were  T h e v a c u o l e s w e r e numerous i n t h e p o s t e r i o r  t h e head  Because  vacuoles  workers r e p o r t e d  e v e n when t h e c o n t e n t  suggest that  a useful  of the perforatorium  function  this  structure  structure  o f t h e acrosome  i n mink  in fertilization.  no c o r r e s p o n d i n g  and i t s  spermatozoa  A l t h o u g h some  I n human  (Fawcett,  1958; B e d f o r d , 1967a;  P e d e r s e n , 1970b),  (Fawcett,  1965) and b a t ( F a w c e t t and I t o , 1965) s p e r m a t o z o a ,  the o c c u r r e n c e o f the p e r f o r a t o r i u m  h a s been  guinea p i g  clearly  demon-  37 strated dog,  i n spermatozoan  cat, rabbit,  1963;  h a r e , and  N i c a n d e r and The  special  Bane,  swellings  anterior  of  the head.  Strong  on  the a n t e r i o r  and  25) where  acid  showed  reported  for rabbit  Bernstein,  1969,  1972,  Flechon  heads  (Nicander  the  rabbit  and  equatorial region  and  and  acrosomal border  and  ring  In  Bane,  while  mammals, t h e i n i t i a l  while the  (PIk6  and  Tyler,  and  B a r r o s and F r a n k l i n  equatorial  region.  spermatozoa  1966;  and  with  part  this  scanning (Ahmad,  Teichman and  hare  and  Teichman,  spermatozoan  of the p o s t a c r o s o m a l Although  the presence of the the a n t e r i o r  i t was  h a r e , o r mink between  Yanagimachi  mammalian  sub-  equatorial  not c o n s p i c u o u s l y  an ovum o c c u r s  of  b o r d e r of the  spermatozoa.  (1968) r e p o r t e d  The  of  been  to the a n t e r i o r  contact  1964;  segment  However, t h e s w e l l i n g s  showed  found i n r a b b i t ,  to a  Bernstein  1974)  in rabbit  spermatozoa,  brane of a spermatozoon region  1972;  the caudal  (1970a)  evident  the s u r f a c e  also  Bane,  1966).  i n conjunction  to that  have  and  Phillips,  swollen  Noda  of hamster  compared  structures  hare are c o n f i n e d  is lightly  o f mink  Bustos-Obregon,  segment,  Yanagimachi  Moreover,  appearance, according  (Nicander  1971;  was  segment  were o b s e r v e d i n t h e  observation  Similar  to the e q u a t o r i a l  b o r d e r s of the e q u a t o r i a l  study.  a swollen  microscopic  boar,  i s the occurrence  phosphatase'activity  the s w e l l i n g s  portion  unpublished).  posterior  posterior  ultrastructural  ram,  g u i n e a p i g (Bane and N i c a n d e r ,  o f mink s p e r m a t o z o a  and  present  electron  from the h o r s e , b u l l ,  1966).  feature  of  (Fig.  heads  large  spermatozoa.  t h e p l a s m a mem-  at the postacrosomal and  Noda,  1970b,c)  i t as o c c u r r i n g  spermatozoa  have  a  at  38 characteristic and  Bane,  specific  structure  1966).  Moreover,  structural  acrosomal  at the e q u a t o r i a l  junction  features which  mechanism d u r i n g  activation  (Koehler,  the e q u a t o r i a l  phatase  activity  useful  i n ensuring  season  i n the mink.  Although  Special  and  in this  be e s s e n t i a l  fertilization  1969).  region  heads  have  of the e q u a t o r i a l  might  attachment  of  spermatozoan  segment ( N i c a n d e r  region  for recognition  and  subsequent  spermatozoa  during  t h e a c r o s o m a l c o n t e n t was  dispersed  for electron  microscopy, the acrosomal  located  the a n t e r i o r  end  intact. the  The  stability  anterior  mammals  part  (Bedford,  Birch-Andersen, 1970b).  1967).  intact  remains  This  stable  during  of or attachment  The  visible  swellings  reported  1966;  region  remained  mammalian stage of  Bane,  Yanagimachi  might  after 1967c;  be  and  Pedersen,  of  1966;  to  i n several  the i n i t i a l  even  be  during  segment  spermaand  spermatozoa B a r r o s e_t a l . ,  useful  i n the  t o an ovum i n t h e i n i t i a l  recogni-  stage of  i n the mink.  p r e s e n c e o f p o s t a c r o s o m a l s h e a t h i n mink  distinct  might  segment compared  been  Bane,  (Bedford,  structure  phos-  and A l m q u i s t , 1964a; Blom  N i c a n d e r and  the corona c e l l s  fertilization  cell  1965;  Saacke  has  d e g e n e r a t i o n ( N i c a n d e r and  penetrate  a  1964;  remains  Noda, 1970a) and  tion  of the e q u a t o r i a l  Furthermore, the e q u a t o r i a l  spermatozoa tozoan  of the e q u a t o r i a l  of the acrosome  egg  a short breeding  processing on  or  modification  c o n t e n t of a c i d  i n mink  fertilization  segment-post-  structural  the high  species-  electron  dense  material  in close  membrane o f t h e p o s t a c r o s o m a l r e g i o n ,  spermatozoa,  apposition conforms  t o the  to the  39 findings  o f Wu  and Newstead  ( 1 9 6 7 a ) and P e d e r s e n tozoa.  The  sheath cell  to adhere  postacrosomal the r e s t it  (1957) c o u l d n o t i d e n t i f y  i n human  while  Although  artifact,  Almquist,  1964a; B e d f o r d ,  confirmed  i t as a normal  The  double  end  of the nucleus  several  bovine  in  this  (Pedersen,  o f mink  cated  neck  i s similar  1972c).  of pores  spermatozoa  o f mink  spermatozoa  the case  prolongation 1965).  1964b).  only at the caudal o f human  S a a c k e and A l m q u i s t  i n t h e n u c l e a r membrane  n u c l e a r pores or i n the study  were  observed  of b u l l  (1965).  was b r i d g e d b y e l e c t r o n  as c o m p l i -  (Fawcett, lies  t h e mink  spermatozoa  has been  the basal plate  dense m a t e r i a l  1970).  w i t h i n the  as shown i n t h e b u l l  p i g spermatozoa  of the basal p l a t e between  i s structurally  the basal p l a t e  In t h i s  of the guinea  The s p a c e  1969a)  spermatozoa.  to that  o f o t h e r mammals  boundary o f t h e i m p l a n t a t i o n f o s s a  from  ( S a a c k e and  Neck  below t h e head,  Almquist,  around  and P h i l l i p s ,  Although  no d e f i n i t e  as i n t h e s p e r m a t o z o a  Immediately  and  investigators  by Blom and B i r c h - A n d e r s e n  The S p e r m a t o z o a n The  found  (1963b) c o n s i d e r e d  f e a t u r e i n mammalian  and t h i s  spermatozoa,  spermatozoa 2.  I-Iadek  n u c l e a r membrane was d i s t i n c t i v e  study  was  to f i t loosely  1965b; F a w c e t t  (1964a) r e p o r t e d t h e p r e s e n c e in  spermatozoan head  i t appeared  o f t h e acrosome.  spermatozoa  a post-  o n l y t o t h e t i p o f t h e a c r o s o m e and t o t h e  sheath  as a f i x a t i o n  sperma-  spermatozoa.  membrane o f t h e mink  firmly  and B e d f o r d  ( 1 9 7 0 b ) i n t h e human and monkey  However, Anberg  acrosomal  (1966) i n t h e b o v i n e  (Saacke  differs  where an a r c u a t e  observed  (Fawcett,  and t h e c a p i t u l u m  as r e p o r t e d by Saacke  40 and  Almquist  observed and  as  (1964a) f o r the  reported f o r Chinese  Phillips,  capitulum  1969b).  was  found  joined  with  fibers  below.  A dorsal  Although denied  capitulum  has  (Fawcett,  1975).  the by  been w e l l  study  or  i n that  with  f i l a m e n t s were  spermatozoa plate  (Fawcett  of  the  the b a s a l p l a t e  above  columns of c o n n e c t i n g  piece  documented not  of  the c a p i t u l u m  Stefanini  the  specific be  spermatozoa  and  i n mature  (1971),  i n mammalian  c o u l d not  of b u l l  fine  ventral  Zamboni and  Whether o r  no  hamster  presence  the f o r m a t i o n of c a p i t u l u m  this  but  and  articulated  the c r o s s s t r i a t e d  s p e r m a t o z o a was  to  bull,  the  spermatozoa  fibers contribute  determined (Saacke  either  and  in  Almquist,  1964b). Following  the c a p i t u l u m ,  c o l u m n s were a r r a n g e d  parallel  axes.  However, b e i n g w i d e s t  nected  ring-like  structure  t h e y were d i v i d e d Finally into  they  two.  into  which  no  had  (1965) and  and  striation  Fawcett  Triplets  of  and  sectioned  of  the  m a j o r and  spermatozoa.  a  con-  clefts.  Distally,  columns.  major column of  long  the  was  split  c o l u m n s were  homogeneously dense m a t r i x m a t e r i a l similar Ito  to those  centriole  and  the normal the o t h e r  demonstrated  (1965) i n t h e  neck r e g i o n .  On  their  formed  minor  cross striations  the p r o x i m a l  or  five  striated  along  four v e r t i c a l  guinea  The  appeared  cutting  angle  to v e r i f y  structural  hand, F a w c e t t  by  p i g and in  p e n t a l e t s ) i n the  m a t e r i a l made i t d i f f i c u l t  were a r t i f a c t s  other  p r o x i m a l l y , they  with  (doublets, t e t r a l e t s ,  sections  to each  four cross  c o l u m n s as e a c h  T h e r e a f t e r , the to s o l i d  forms  two  became n i n e  connected  at l e a s t  of  bat.  varied  sagittal the  i f these  variation  Fawcett  forms  i n mink  (1958) m e n t i o n e d  that  41 the  proximal  different  centriole  from  occurrence  the t y p i c a l  of the c e n t r a l  seem t o be r e m n a n t s of  o f mammalian centriole pair  t h e mink  of the c e n t r i o l e  i s probably  microtubules  i n t h e neck  and  (1969b)  Phillips  pair  i n the connecting region  of a somatic c e l l .  triplets  region that  piece  spermatozoa.  to  t h o s e o f t h e b a t , dormouse, R u s s i a n hamster boar  3.  The S p e r m a t o z o a n Unlike  siderably mink  and Bane,  i s similar  variation.  tail  follows  The a x i a l  the usual  1961;  F a w c e t t a n d I t o , 1965; F a w c e t t , 1 9 7 0 ) .  appeared  slightly  observation  of microtubules, larger  (1970,  Warner and S a t i r  (1973)  spermatozoa r e p o r t e d outer  arm o f s u b f i b e r  distally, arm,  regard  that  subfiber  A which  agrees with  In g e n e r a l ,  (Telkka  e t a_l. ,  In t h e a x i a l  (Afzelius,  1959).  water  B was l a r g e r  the f i n d i n g s  earlier How-  t o mammalian, and  bore a f l a g - l i k e  the s t r u c t u r e  bundle of pattern  agrees with  i n the case o f f r e s h  a p p e a r e d t o be s t r u c t u r a l l y  and t h i s  (1973).  spermatozoa  1975) w i t h  fiber  shows c o n -  the diameter of subfiber A  t h a n B, w h i c h  o f sea urchin  ever, Fawcett  The s c r o l l  ( F a w c e t t , 1970)  9 + 9 + 2  i s commonly o b s e r v e d i n o t h e r mammals  complex  Fawcett  i n structure  of the spermatozoon  which  filament  have  1962b).  the t a i l  species  spermatozoan  which  Tail  t h e head,  less  suggests  had t h e c e n t r a l  t h e neck  and  spermatozoa  which  spermatozoa.  some s p e c i e s  o f matured  piece,  species  in  (Nicander  o f mink  o f matured  The  or the extension  of the connecting  one o f t h e s p e c i a l  stated  was n o t  and d o u b l e m i c r o t u b u l e s ,  t h e axoneme i n t h e i n t e r i o r  that  spermatozoon  different  mussel  t h a n A.  The  projection from  the inner  o f Warner a n d S a t i r  of microtubules,  radial  42 spokes to  and  that  spermatozoa  the middle p i e c e  doublets  the  s h e a t h o f mink  o f mammalian  In  dense  central  resemble  fibers  appear  than  fibers  the r e s t  t o be  (number  o f the dense  differs  from  a usual  phenomenon, t h r e e  (1964b)  and  the r e s u l t  fibers  thicker  in bull  b r e e d s o f t h e same s p e c i e s .  four  larger  reported  larger dense  spermatozoa.  than  distal  of the t a i l  sheath which chondrial in  g y r e s i n mink  and need  a single  with species  the other  normal  dog,  suni,  longer  also  and  dense  Therefore,  t o end  long The  long  i n the fibers.  mitochondrial number o f m i t o than  chinchilla,  Because  the  of the dense  i s greater  period  but  i n mammalian  appeared  and  between  Wu  four  R u s s i a n hamster,  (Fawcett, 1970).  for their  hand,  of the f i b e r s ,  helix.  spermatozoa  and A l m q u i s t  difference  sooner than the r e s t  than i n t h e dormouse,  more e n e r g y  Saacke  a comparatively  as  1, 5, and 6) and s i x  i n the b u l l .  8)  but  described,  o r sometimes  t o be  3 and  o f human, b u l l ,  r a t spermatozoa  three  the length  had  seems t o be  spermatozoa  less  seem  (number  spermatozoa  (1955) who  not o n l y On  In d i a m e t e r  i n the b u l l ,  (number  the o t h e r s  Considering fibers  Mink  that  fibers  a x e s o f two part  varied  of  i s consonant with the  the comparative s i z e  fibers  outer  to those of  6) were l a r g e r  spermatozoa.  with  were  the nine  Furthermore, four  This  of B r a d f i e l d  of the dense  fibers  and  fibers.  each  (1966)  spermatozoa,  B i r c h - A n d e r s e n (1960)  suggested that  Newstead  similar  1966).  s m a l l e r when compared  9, 1, 5,  o f Blom  dense  o f mink  (Fawcett, 1970).  finding  thiner  (Hancock,  appear  t h o s e o f o t h e r mammals, b u t t h e n i n e  lower v e r t e b r a t e s  the dense  region  spermatozoa  mink  those found but  i t is  mouse, b a t , spermatozoa  of e x i s t e n c e  i n the  43 uterine that  tract  of the female  of other  the middle p i e c e  ejaculated Bishop  it  bull  cytoplasmic  i n some mink  droplets  triangular  mammalian  a n n u l u s was  p i g ( F a w c e t t , 1965,  ( S a a c k e and A l m q u i s t ,  the o t h e r  on  the a n n u l u s , which  of  Saacke An  spermatozoa  present  nal mink not with  columns  1967a). i n mink  regard  pattern piece,  o f t h e axoneme w h i l e Anberg  pattern  reveals  present no  This  denied  posterior  arrangement  even  However,  though  The  maintained i n the  (1957) r e p o r t e d piece  a possible i n human  loss  s t u d y o f mink in structure  segment  piece,  characteristic  they are  from  the modified  o f mink  basic end of 9 + 2  spermatozoa  o f t h e head  columns,  those  spermatozoa.  the occurrence of s w e l l i n g s  of the c o n n e c t i n g  longitudi-  i s i n accordance with  was  t o the e q u a t o r i a l  features  columns  (9+2)  the g r o u p i n g of the s t r i a t e d  important  of  (Anberg, 1957).  (Fawcett, 1970).  ultrastructural  the. f i n d i n g s  the e x i s t e n c e  finding  itself  spermatozoa.  mammals  Hov/ever,  antelope,  to anchor  consonant with  fundamental d i f f e r e n c e  o t h e r mammals.  and  membrane a p p e a r e d  somewhere i n t h e end  The  suni  as  ( P e d e r s e n , 1970b).  longitudinal  to several  spermatozoa  man  i s closely  very conspicuous.  1957a;  1964b),  investigator  had  (Hancock,  (Andersen, 1974),  i n the f i b r o u s .sheath  spermatozoa  were f o u n d  fox  A l m q u i s t (1964b) i n b u l l  earlier  larger  1970), b l u e  hand, the c e l l  and  a  with  s p e r m a t o z o a as i n o t h e r  1966a; O r g e b i n - C r i s t ,  On  and  as c o m p a r e d  i s i n t h e b a t , dormouse, C h i n e s e hamster,  guinea  and  The  or epididyrnal  and W a l t o n ,  The  1952)  domestic a n i m a l s , they might r e q u i r e  number o f m i t o c h o n d r i a . on  (Enders,  those of anterior and  the  capitulum  a r e some o f t h e spermatozoa.  44 F.  SUMMARY  The  subcellular  obtained tron  from  s t a n d a r d dark  microscopy, The  structure  dense n u c l e u s w i t h  head,  f l a t t e n e d and  thirds  the  some may  one-third be  divided  by  mostly  in outline.  covered  means o f  elec-  techniques. o f an  o f v a c u o l e s , was  ovate  n u c l e u s was  spermatozoa  studied  composed  a number  ventrally  posterior  mink was  using thin-sectioning  spermatozoan  of  of epididyrnal  with  electron  seen  The  t o be  anterior  the acrosome  with  the  postacrosomal  sheath.  into  the  apical,  and  main,  dorsotwo-  and  the  The  acro-  equatorial  segments a c c o r d i n g t o t h e a c r o s o m a l c o n t e n t a r o u n d t h e n u c l e u s . On  the  t i p of  the  nucleus,  t h e p e r f o r a t o r i u m was  remained  i n t a c t e v e n when t h e c o n t e n t  lodged.  On  swellings side  the d o r s a l  were o b s e r v e d ;  of  the  was  associated  The  postacrosomal aspect  acrosomal cell  and  to the  with  the  sheath  o f t h e s e were  the c e l l  of  the  a dense  membrane.  postacrosomal from  the r e s t  accommodated  firmly  The  to the  of  the  at  on  each  d e p o s i t e d on and  the a n t e r i o r t i p of  was  acrosome.  the c a p i t u l u m of  each  segment,  equatorial  than  found The  dis-  head, s i x  located  region  layer  sheath, but  the  equatorial  postacrosomal  was  membrane a d h e r e d  the nucleus the  two  and  t h e a c r o s o m e was  aspects of  s e g m e n t s were more s t a b l e  The  separated  of  ventral  proximal border  one  inner  and  of  visible  the  side. the  postpart.  acrosome  t o be  usually  caudal  t h e neck  surface  by  forming  implantation fossa. The  articular  separate dorsal  and  structure ventral  of  the  plates  neck of  appeared  and  to  the c a p i t u l u m  show which  of  45 were f o l l o w e d by piece. five  These  a ring  striated  of  surface  of  of  the  the c e n t e r  of  the  appeared  axial  fiber  capitulum,  the  the connected  axoneme were o c c a s i o n a l l y column b u n d l e . found  i n the The  the  A few  spermatozoan  dense f i b e r s , dense f i b e r s  tail  the  double  s u b f i b e r A was  that of  was  o f medium  The  annulus  9+2  Beneath  the the  was  remnants of  i n the and  interior  scrolls  major  and  nine inner  found  in  the of  were  the also  displayed a 9 + 9 + 2 pattern in  number 9, 1, 5,  of  dense f i b e r s .  triangular  caudal  and  In t h e  the  l e n g t h compared  bundle  larger  of  the  smallest. other  The  i n diameter  distributed  central The  sections  electron  the  piece  spermatozoa. and  dense  some s p e r m a t o z o a ,  c y t o p l a s m i c d r o p l e t s were  end  piece.  The  t h e end  piece maintained  of  columns.  the  middle  Although  p a t t e r n , i t d i d not  have the  fibrous  than  fiber,  middle  mammalian  in longitudinal  the  pair.  axonemal complex,  than  with  consisting  a central  6 were  larger  non-homogeneously In  fiber  and  s u b f i b e r B was  was  longitudinal  two  centriole  The  microtubules,  diameter  the  proximal  ring.  axial  the  on  bundles.  mitochondria  of  material.  connecting  to continue with  visible  the r e s t  contained  the  neck r e g i o n .  o r g a n i z a t i o n of  while  columns of  c o l u m n s were f o l l o w e d by  minor columns which  dense f i b e r s  striated  fibrous  sheath.  found  sheath  had  the  46 Chapter  II  CYTOCHEMICAL OBSERVATIONS OF  A.  problem  of spermatozoa  in reproductive  i n the m o t i l i t y ATP  tozoa,  and  either  glycolysis  other  spermatozoa. the  and  i s the energy  energy of f r u c t o s e  i n s e m i n a l plasma,  nases,  and  enzymes  pathway o f g l y c o l y s i s Related sheath  of  of the middle p i e c e  motility.  Several  in  The  sperma-  coenzymes,  by  i n the  hydrolyses  known ATP  as  and  dehydroge-  i n t h e Embden-Meyerhof  tricarboxylic  localized and  i s stored  doublets,  aerobic  enzymes a r e m o s t l y  of  spermatozoa.  of spermatozoa,  are involved  and  penetration  phospholipid  and ATP  i n the f l a g e l l a  for motility.  other  trans-  i n semen a r e t r a n s f o r m e d t o ATP  or r e s p i r a t i o n  ATPase  energy  action  source of spermatozoan  substrates  i s the  Enzymes p l a y an e s s e n t i a l  i n the l y t i c  d y n e i n arms o f t h e a x o n e m a l  releases  physiology  t o t h e egg w i t h s u b s e q u e n t  c u m u l u s c e l l s and membranes o f t h e e g g . role  SPERMATOZOA  INTRODUCTION A basic  port  MINK  i n the  acid  cycle.  mitochondrial  the p r i n c i p a l  piece  of  sperma-  tozoa. The form  egg  i s s u r r o u n d e d by  several  the z y g o t e , the spermatozoon  ment o f t h e ovum by Hyaluronidase  and  the l y t i c  acid  many a c r o s o m a l enzymes. transport  of phqsphate  spermatozoa.  must  action  phosphatase  protective penetrate  of  the  the acrosomal  To  investenzyme.  a r e i m p o r t a n t enzymes among  Some p h o s p h a t a s e s groups  coats.  between  are i n v o l v e d  s e m i n a l plasma  and  i n the  47 Although between  the a c t i v i t y  convincing  i n v e s t i g a t o r s have  i n d i f f e r e n t species  i t might Although  there  on t h e enzyme  Enzyme  t h e enzyme a c t i v i t y o f  localization  m i g h t be h e l p f u l  have  as an i n d e x been  several  o f mammalian  o f spermato-  to e l u c i d a t e the  to predict studies  spermatozoa,  i n mink s p e r m a t o z o a .  present  study  enzymes  (phosphatases, esterase,  in  was  the r e l a t i o n  m e t a b o l i s m and f e r t i l i z a t i o n  a l s o be u s e d  enzyme l o c a l i z a t i o n report  Moreover,  among s p e c i e s .  nature of spermatozoan and  studied  o f some enzymes and t h e semen q u a l i t y ,  r e s u l t s a r e few.  semen d i f f e r s soa  several  to i d e n t i f y  fertility.  regarding there  the  i s no  The p u r p o s e o f t h e  the l o c a l i z a t i o n oxidase,  process,  sites  o f 14  and d e h y d r o g e n a s e s )  mink s p e r m a t o z o a and t h e i m p l i c a t i o n o f t h e s e enzymes i n  regards  to the reproductive  physiology  o f the mink.  48 B.  REVIEW OF  1.  Early  RELATED  Studies  Although seminal on  smell  1786,  and  one  Miescher and  of Spermatozoan C y t o c h e m i s t r y  early  plasma  LITERATURE  investigators  as a v e h i c l e  taste  century (1897)  d i d not e n t i r e l y  (Leeuwenhoek,  o f semen were f i r s t after  the d i s c o v e r y  gave much  seminal plasma.  information  However, u n t i l  1677),  reported  neglect  observations by H u n t e r i n  of spermatozoa. concerning  Ivanov  (1907)  succeeded i n  from d o m e s t i c a n i m a l s f o r a r t i f i c i a l  tion,  workers  spermatozoa In  and  1919  nutrients  Walton  tozoa could  survive  (1927)  Redenz  the  and and  lactic  carbon  et. al_. under  showed  extra-cellular  aerobic  mussel  1933;  or  after  (1928)  fully  found that  anaerobic  differentiated.  mammalian  conditions,  receive  sperma-  and M c C a r t h y e_t  i n human semen.  Later  occurring  i n the seminal plasma, i . e .  and  utilize  through g l y c o l y t i c  breakdown u n d e r  conditions.  aerobic  p r o d u c e d by  Under  glycolysis  i s further  both  conditions, degraded  to  water.  i s provided  action by  1956).  factor  on  t h e egg membrane o f t h e  the spermatozoan Hoffman  (1931) d i s c o v e r e d  "diffusing"  being  not  substrates  Wada e t a l . ,  and M c C l e a n  spermatozoa could  spermatozoa c o u l d  anaerobic  dioxide  invertebrate  mammalian  glucose,  acid  with  that  A powerful solvent and  that  demonstrated g l y c o l y s i s  (1933)  fructose  stated  seminal plasma  Afterwards,  al.  to deal  insemina-  semen.  Lillie  from  used  Later,  spermatozoa  semen c o l l e c t i o n most e a r l y  the  and  Duran-Reynals  the e x i s t e n c e  i n mammalian  acrosome  testes.  of a  toad  (Parat, (1931)  "spreading"  The d e p o l y m e r i z a -  49 tion  and h y d r o l y s i s  reported  from r a b b i t  2.  (1955).  Spermatozoan  spermatozoa d i s p e r s e  oophorus  of the r a b b i t  The R o l e o f Enzymes  The ATP  acid  by h y a l u r o n i d a s e  by M e y e r e t a l . ( 1 9 3 7 ) , M c C l e a n  and Weissmann  cumulus  of hyaluronic  and R o w l a n d s  the f o l l i c l e  cell  i n Spermatozoan  Motility  source of spermatozoan  i n spermatozoa  (Lardy  and P h i l l i p s ,  1945a,b,c). initiates  Mammalian  are depleted  However,  (Lardy  d e c r e a s e o f ATP i s r a p i d  the  loss  lose  1945a;  their  and P h i l l i p s ,  spermatozoan  movement when ATP i s added  motility i s  1945; Mann,  spermatozoa  the i s o l a t e d  the  of the  o v a (Yamane, 1930, 1 9 3 5 b ) .  energy  when ATP r e s e r v e s  (1942)  suspensions or extracts  immediate  I v a n o v et. _ a l . 1 9 4 6 ) .  were  (Bishop,  o f ATP i s d e c r e a s e d by t h e a d d i t i o n  1945; Mann,  tail  model  1958).  i n the absence of a  motility  While  substrate,  of glucose or  fructose  (Lardy  and P h i l l i p s ,  1945; Mann, 1 9 4 5 a ) ,  and by p h o s -  pholipid  (Lardy  and P h i l l i p s ,  1945).  phosphate of  ATP  i s furnished  1964; ATP  B i s h o p and W a l t o n ,  by t h e a c t i o n  nemal  by g l y c o l y s i s  doublets  of the t a i l  in  systems, the major  mammalian s p e r m a t o z o a  anaerobic energy  Actually,  (Gonse,  are species  o f ATPase  from  ( G i b b o n s and  differences  e n e r g y s o u r c e f o r ATP  i n energy generation  seminal plasma f o r  1962; Mann,  1964);  phospholipid  (Lardy  the secondary and  Phillips,  1945; L a r d y e t a l . , 1 9 4 5 a ; H a r t r e e and Mann, spermatozoa use g l y c o l y t i c  (Mann,  t h e arms o f t h e a x o -  i s fructose-rich  s o u r c e i s endogenous  1941a,b, Because  glycolysis  metabolism  The e n e r g y i s r e l e a s e d  a r e composed  Rowe, 1965).- A l t h o u g h t h e r e acquiring  or oxidative  1966a).  of ATPase.  Energy-rich  phosphorylation  1961).  preferen-  50 tially, 1962),  even  under  aerobic  the m e t a b o l i c  conditions  pattern  of spermatozoa  c a n be r e g a r d e d as a m e t a b o l i c requirements glycolytic  for survival  degradation  accomplished c; F l i p s e , 1959;  1954,  Johnson,  the  substrate  pyruvic  acid,  and  which  and  (Mann,  The  (Mann,  1955,  Wu  1945b, eta l . ,  Kayne,  1975).  in this  pathway  1945b,  1964).  and  Finally,  dephosphorylated to produce  i s converted  to l a c t i c  acid  i n the  absence  oxygen. In  the p r e s e n c e of oxygen,  considerable lactate,  respiratory  1948; The  (Lardy  cycle  and  activity catalytic  Mammalian  1941a,b, 1945) 1957)  1957;  and by  amino a c i d s  (Nelson,  ATP  1946), c y t o c h r o m e s 1954,  1958;  Lutwak-Mann, 1973b).  tricarboxylic  of the cytochrome  lipid  The  (Lardy  enzyme s y s t e m specific  a_l. , 1945a;  (Mann,  Mann, 1964;  and  1956b; F l i p s e  includes et  system.  metabolize glycerol  (Flipse,  (Lardy  sugars,  and  Johnson,  the- K r e b s  1957),  of spermatozoa  enzymes:  Ivanov e t a l . , ATPase  activity  on  the Krebs c y c l e .  the r e s p i r a t i o n several  depends  White,  Mann and  T u r n e r and  show  glycolysable  butyrate,  1945;  spermatozoa can a l s o  (Mann and W h i t e ,  Benson,  1953;  spermatozoa  consuming  propionate,  Phillips,  M e l r o s e and T e r n e r ,  respiratory  acid  and  mammalian  activity  pyruvate, acetate,  oxaloacetate  and  Storey  special  spermatozoa i s  scheme  and A l m q u i s t ,  spermatozoa  i s hydrated  i s u n u s u a l and i t  i n mammalian  1973a;  Lodge,  of m o t i l i t y .  enzymes and c o e n z y m e s a r e i n v o l v e d  found i n i n t a c t  in  Flipse  and  to the  with maintenance  of hexose  1956a;  are  of  adaptation  t h r o u g h t h e Embden-Meyerhof  T u r n e r and  Several  (Salisbury  U e s u g i and  and involved  coenzymes  Mann,  1945a,c,  Phillips,  1945a,c:  1951), Yamazoe,  51 1966;  Quinn  1959;  Salisbury  1965;  Harrison  cytochrome  and  White,  and Lodge,. 1962; and W h i t e ,  oxidase  confined Walton, in  tail  while  the r e s p i r a t o r y  On  (Mann,  hand,  1941a,c)  species.  Therefore,  et a l . ,  also  R o l e o f Enzymes  1962)  differ  in  tract  (Austin,  energy  s o u r c e s i n the environment  acrosomal digests oophorus 1974). the et  reaction  distribution  The  cementing  ovum i s d i g e s t e d a l . , 1969a;  acid  1951;  Chang,  ( R o g e r s and labilized  between  Rowlands,  and  among  of  enzymes  Talbot  Williams,  by  The  the types of  Yanagimachi, and  of the and  1970)  Franklin,  enzymes which  which  cumulus  the corona c e l l s  corona-penetrating  Z a n e v e l d and  i n the  1951).  plasma  the c e l l s  1942;  the  of hyaluronidase,  s u b s t a n c e between by  (Lardy  species.  membranes e n a b l e t h e r e l e a s e  ( M c C l e a n and  glycolysis  substrates  i s influenced  the c a p a c i t a t i o n ,  the h y a l u r o n i c  exist  processes  t h e ovum t h r o u g h c a p a c i t a t i o n  of t h e acrosome  Through  of  s p e r m a t o z o a must a c q u i r e  timing  1975).  enzymes  are d i f f e r e n t  among  and  Fertilization  to f e r t i l i z a t i o n  to penetrate  effects  and v a r i o u s  t o be  (Bishop  1941c), o x i d a t i v e  t h e p r e s e n c e and  spermatozoa might  female r e p r o d u c t i v e  The  and  through the  seem  some m e t a b l i c  1967).  and P h i l l i p s ,  1941a,b; S c o t t  enzymes  of the middle p i e c e  the o t h e r  Phillips,  ability  1975),  distributed  Phillips,  Prior  Kayne,  t o be  (Lardy  The  and  enzymes a p p e a r  (Lardy  3.  Storey  1955,  Mohri eta l . ,  1958).  the s e m i n a l plasma  and  G o l d b e r g , 1963;  1955,  to the mitochondria 1966b).  (Nelson,  1954,  inhibitors  in  1974;  (Nelson,  The m e t a b o l i c spermatozoan  1968), dehydrogenases  of  (Zaneveld have  been  recently  classified  Proteolytic  as e s t e r a s e s  enzymes on  membrane a r e e i t h e r state  (Srivastava  ovum i s d i g e s t e d  the inner  gradually  et a l . , through  enzymes  1973).  crude e x t r a c t  the  vitelline  tration  therefore, lytic  released  1974).  (Srivastava  membrane,  (Srivastava  surface  A  t h e zona  proteolytic The  (Bradford  or a c t i v a t e d  limited  o f the acrosome barrier  plays  i n a bound  fissure by  et. _ a l . , 1965;  the l a s t  1976a,b).  of the acrosomal  pellucida  e t a_l. , 1 9 6 5 ) .  t h e acrosome  et a l . ,  an  of  the  array  of  Z a n e v e l d et. a l . ,  frequently  digest  to spermatozoan  During  pene-  fertilization,  an e s s e n t i a l  role  to  provide  agents. Owing  treatments  t o the improvement  t o remove t h e a c r o s o m e  numerous l y t i c  enzymes have  These  are:  1965;  Bernstein  resemble  of p h y s i c a l  hyaluronidase  been  p l a s m i n and  o f mammalian localized  (Rowlands,  and T e i c h m a n , trypsin  1973),  (Yamane,  Stambaugh  (Wislocki,  1950;  (Bernstein  and  (Conchie  and  Teichman,  Mann, 1957;  d-mannosidase ( B r y a n and  Bernstein  and  1973),  Allison  U n n i t h a n , 1972,  and  1973;  neuramidinase,  and  1975).  Teich-  phosphatase  1973)^ p - g l u c u r o n i d a s e  Hartree,  ( M c R o r i e and  1970),  non-specific  Bradford  ( G a r b e r s e t a l . , 1970;  et a l . ,  and  et  1973), p - N - a c e t y l g l u c o s a m i n i d a s e  5-nucleotidase  Multamaki  enzymes t h a t  Bernstein  acid  Teichman,  ( C o n c h i e and Mann, 1 9 5 7 ) ,  others  eta l . ,  1935a,b; S r i v a s t a v a  man,  Smith,  acrosome.  Srivastava  proteolytic  Z a n e v e l d e_t a_l. , 1969b, 1973; and  spermatozoa,  i n the  1944;  a l . , 1965; 1973;  and c h e m i c a l  et a l . ,  Multamaki Williams,  esterase  1976a,b),  e t a_l. , 1974;  1975),  Some p h o s p h a t a s e  activities  (Melarnpy e t _ a l . , 1952; and  Hartree,  Quinn  and Wh-ite,  activity Acid  1970;  Wislocki,  and  Mathur,  pyrophosphatase, contained  and  most  several  as  spermatozoa  collected  mature  and  immature  fertile  Orgegin-Crist, difference lated  Epididymal exchange 1942; to  of metabolic pattern has b e e n  spermatozoa  compared  1942).  spermatozoa  Moreover,  produce  than under  aerobic  glycolysis  rate  absence  lactic  and  1962),  active  are  also  Study  mammals  several  (Young,  1972;  1931; The ejacu-  investigators. respiratory  ( H e n l e and  the a d d i t i o n oxygen  t o be  e t a_l. ,  e p i d i d y m a l and  uptake  Zittle,  of glucose ( H e n l e and  of sugar, e p i d i d y m a l  more r a p i d l y  under a n a e r o b i c  In e j a c u l a t e d  1964).  the  appear  (Pomytko  spermatozoa  i s n o t much h i g h e r (Mann,  ATP  B e d f o r d , 1972).  by  addition  conditions.  o f oxygen  split  Lodge,  between  stimulates  acid  1964).  5-nucleotidase,  epididymis  However,  on  (Mann,  a r e t h e most  a lower endogenous  to e j a c u l a t e d  e p i d i d y m a l spermatozoa  and  reported  have  phosphatase  f o r t h e Enzyme  i n other  Horan  L a r d y e_t a_l. , 1 9 4 5 b ) .  Zittle,  the  1968;  1966;  1964).  i n t h e mink  also  Allison  consider epididymal  the cauda  not o n l y  1967a,  spermatozoa  (Mann,  as a M o d e l  from  powerful  enzymes w h i c h  (Salisbury  Ahmad e_t aJL. , 1975b) b u t  and  phosphatase  investigators  spermatozoa  1969;  U e s u g i and Yamazoe,  1971),  i n the seminal plasma  Although  Bernstein,  i n t h e semen.  E p i d i d y m a l Spermatozoa  i n spermatozoa  i n the s e m i n a l plasma  alkaline  enzymes  and  1950;  o f t h e semen e x i s t s  dephosphorylating  4.  Teichman  1968;  phosphatase  are detected  spermatozoa  i n the presence than i n Washed  ejaculated  the  54 spermatozoa extent  On  the c o n t r a r y , and  Pasteur  effect  basic  Murdoch  ejaculated  in  1962).  testicular  used  Kayne,  spermatozoa  Gonse,  al.,  and  (1968)  1975). i s also  Moreover,  fluid  change  spermatozoa  1967).  The  i . e . the  compared  Phillips,  and Wales  White  to  1941a,b,c,  1943a,  (1961), Voglmayr  et a l .  reported  no d i f f e r e n c e i n  ejaculated  i n cauda e p i d i d y m a l  pathway  major  spermatozoa.  (Wu  energy  e_t _al_. ,  source of  testicular  spermatozoa  metabolic pattern  (Lardy  (Mann,  stored  toward  that  e_t a_l. , 1945b; V o g l m a y r  Consequently, e p i d i d y m a l spermatozoa  of spermatozoa  fresh  v i a anaerobic glycolysis  their  as a c o n v e n i e n t model  bolism  and  b e t w e e n e p i d i d y m a l and  and  of e j a c u l a t e d et  White  by o x y g e n ,  i s v i a t h e Embden-Meyerhof  Storey  1964;  (Lardy  Pasteur effect,  mechanism o f g l u c o s e u t i l i z a t i o n  spermatozoa 1959;  a feeble  inhibition  spermatozoa  (1966),  The  only  of g l y c o l y s i s  epididymal b).  exhibit  have  been  f o r t h e d e m o n s t r a t i o n o f t h e meta-  which  might  be  applied  for ejaculated  samples. Furthermore, spermatozoa et  al.,  from  1973),  (Zaneveld  a proteinase seminal plasma  and  et a l . ,  phosphatase  is later 1971,  activity  Acrosin  passway  f o r spermatozoa  than If  in ejaculated  such i n h i b i t i n g  seminal  plasma  and  lost  1973).  activity,  higher  during after  ejaculation  origin  inhibits  spermatozoa  in. u t e r o the  ( S i v a r a m and  acrosomal p r o t e i n a s e  i n e p i d i d y m a l and  by  (Zaneveld  capacitation  through the zona  rabbit  i s absorbed  L-tartrate  of p r o s t a t i c  1971).  significantly  inhibitor  capacitated (Zaneveld  factors  of other  inhibit  t h e enzyme a c t i v i t y  Bami,  digesting  pellucida,  acid  a  is spermatozoa  e_t a l _ . , 1 9 6 9 b ) .  enzymes e x i s t of  i n the ejaculated  55 spermatozoa,  epididymal  for  the r e a l  studying  spermatozoa  distribution  might of  be a good  enzymes.  model  56 C.  MATERIALS .For  AND  the cytochemical  cauda e p i d i d y m i s saline the  solution  tissue.  suspension and  METHODS  were i m m e d i a t e l y where  Motile were u s e d  small  pieces  spermatozoa  of the  immersed i n 1 ml o f  s p e r m a t o z o a were r e l e a s e d by thus  f o r preparation  s t a i n e d f o r enzyme  Table  investigation  released  into  of microscope  0.85%  squeezing the slides  c h a r a c t e r i z a t i o n as i n d i c a t e d i n  1. All  t h e s u b s t r a t e s were o b t a i n e d  from  Sigma C h e m i c a l  Company.  The s t a i n e d sperm p r e p a r a t i o n s  were mounted i n  Farrant's  medium  microscope.  and e x a m i n e d by a l i g h t  p r e s e n c e o f t h e enzymes was  confirmed  staining  characteristics  prepared  by t h e d e l e t i o n o f s u b s t r a t e  inhibitors.  by c o m p a r i s o n  of appropriate  control  The with  the  slides  o r by t h e a d d i t i o n o f  Table  1  THE METHODS OF ENZYME  Method  Enzyme  37 C,  Glucose-6-phosphatase ADPase  Lead  method  37 C, 20 min  Lead  method  37 C, 1 h  ATPase  Calcium-activated method  3 7 ° C , 20 min  Non-specific esterase DOPA o x i d a s e  Indoxyl method  3 7°C  30 min  3 7°C,  2h  A l k a l i n e phosphatase 5-nucleotidase  Malate dehydrogenase Succinate dehydrogenase Lactate dehydrogenase Isocitrate dehydrogenase 6-phosphogluconic dehydrogenase NADH d i a p h o r a s e  Diengdoh  acetate method  NAD and MTT N i t r o BT NAD and MTT NAD and MTT NADP and MTT NAD and N i t r o BT  U  2h  37°C, l h 37°C,  20 min  Room temp, 1 h 3 7°C, l h Room temp, 1 h Room temp, 1 h Room temp, 1 h Room temp, 30 m i n  Reference  Control  Incubation  Gomori l e a d phosp h a t e method Gomori-Takamatsu procedure L e a d method  A c i d phosphatase  LOCALIZATION  A d d i t i o n o f 0.01M sodium f l u o r i d e Deletion of the substrate The s u b s t r a t e was r e p l a c e d by s o dium-B-glycerophosphate - ditto Deletion of the substrate The s u b s t r a t e was r e p l a c e d by s o dium^- glycerophosphate D e l e t i o n of the substrate A d d i t i o n o f potassium cyanide Deletion of the substrate - ditto -  ditto  -  Addition of sodium c y a n i d e - ditto D e l e t i o n of the substrate  C h a y e n e t a l . (1973) pp. 112-114 C h a y e n e t a l . (1973) pp. 108-109 C h a y e n e t a l . (1973) p. 118 C h a y e n e t a l . (1973) pp. 120-121 W a c h s t e i n and Meisel (1957) C h a y e n e t a l . (1973) p. 124  C h a y e n e t a l . (1973) pp. 137-139 C h a y e n e t a l . (1973) pp. 170-171 P e a r s e (1972) pp. 1343-1344 N a c h l a s e t a l . (1957) P e a r s e (1972) pp. 1343-1344 - ditto -  ditto  -  N a c h l a s e t a l . (1958)  58 D.  RESULTS  1.  Phosphatases  Acid localized region  phosphatase  among t h e e x a m i n e d  o f t h e head  were d e e p l y head  ment.  activity  stained.  middle  piece  activity  was  v/as f o u n d  ( F i g . 29).  sheath  w h i l e v/eak a c t i v i t y  phosphatase stained 2.  E s t e r a s e and  base  a p p e a r i n g as two  sheath activity  capitis,  v/as w e a k l y  localized  glucose-6-phos-  was c o n f i n e d t o t h e  diphosphatase strongly  (ADPase,  i n the t a i l  while  postacrosomal  t r i p h o s p h a t a s e (ATPase, i n t h e head  v/as d e t e c t e d i n t h e g a l e a sheath  seg-  ( F i g . 2 6 ) . • The  i n t h e a c r o s o m e and  strongly  i n the  (EC 3.1.3.1)  strong  activity  piece)  ( F i g . 30).  base  EC  and  3.6.1.3) tail  capitis, A l l the  localized  by t h e p r e s e n t t e c h n i q u e  were  i n colour.  Non-specific the  Very  complexes determined  brown  equatorial  that  i n the g a l e a  distributed  acrosome and p o s t a c r o s o m a l  The  and p o s t a c r o s o m a l  and t a i l  Adenosine  Adenosine  v/as d i s t r i b u t e d  strongly  o f the e q u a t o r i a l  phosphatase  ( F i g . 27).  ( F i g . 28).  weak a c t i v i t y  activity  border  acrosome,  ( G - 6 - P a s e , EC 3.1.3.9) piece  noticed  prominances  o f 5 - n u c l e o t i d a s e (EC 3.1.3.5)  EC 3.6.1.5)  sheath  stained  Alkaline  postacrosomal  was most  ( F i g . 25).  especially  and e v e n l y d i s t r i b u t e d  the middle  phatase  enzymes  humps on t h e a n t e r i o r  strongly  acrosome,  in  two d e e p l y  activity  ( m i d d l e p i e c e and p r i n c i p a l  I t was  The g a l e a c a p i t i s ,  were a l s o was  and t a i l  stained.  t h e r e were  connected  (EC 3.1.3.2)  Oxidase esterase activity  o f t h e head  and t h e m i d d l e  v/as w e a k l y piece  localized in  ( F i g . 31).  59 3,4-dihydroxyphenylalanine demonstrated faintly tail  as c a t e c h o l  present  3.  oxidase,  i n the acrosome,  ( F i g . 32).  stained  (DOPA) o x i d a s e  The e s t e r a s e  light-grey  EC 1.14.18.1) a c t i v i t y  was  p o s t a c r o s o m a l s h e a t h , and  and o x i d a s e c o m p l e x e s  and i n t e n s e - g r e y  were  respectively.  Dehydrogenases  Most  of the dehydrogenases  the m i d d l e p i e c e , grey. 99.1)  and t h e i r  The r e a c t i o n  was  dehydrogenase  examined  reaction  of succinate  and r e d u c e d n i c o t i n a m i d e  diaphorase  strong  (Figs.  were c o n f i n e d  p r o d u c t s were  dehydrogenase  34,37),  and t h a t  (SDH, EC 1.3.  of  (LDH, EC 1.1.1.27) and i s o c i t r a t e  activity  o f malate dehydrogenase  detected  as b e i n g  b a s e o f t h e head  strong  (MDH,  (Figs.  (NADH)  lactate dehydrogenase 35,36).  and p r i n c i p a l  piece  The  EC 1.1.1.37)- was  i n t h e m i d d l e p i e c e and weak  6-phosphogluconic dehydrogenase undetectable.  weak  only to  stained  adenine d i n u c l e o t i d e  (IDH, EC 1.1.1.41) was c o m p a r a t i v e l y  was  (histochemically  i n the  ( F i g . 33) w h i l e  (PDH, EC 1.1.1.43)  activity  60 Figures  24 t o 37.  spermatozoa.  Enzyme  24.  Control  Figure  25.  Very  localized  26.  and  tail.  27.  i s especially  the middle 28.  29.  30.  Very  weakly  strong  acrosome,  prominent.  The  sheath are  31.  localized  and w e a k l y  i n the  sheath.  triphosphatase  strongly  activity i s  activity i s  s t r o n g l y i n the t a i l  i n t h e head capitis,  activity i s base  and t a i l ,  and  acrosome and p o s t -  sheath.  Non-specific and m i d d l e  e s t e r a s e a c t i v i t y c a n be s e e n i n t h e piece.  DOPA o x i d a s e a c t i v i t y  acrosome,  i s weakly  sheath  piece.  diphosphatase  i n the galea  base  32.  postacrosomal  glucose-6-phosphatase  to the middle  Adenosine  acrosomal  Figure  of the  piece.  Adenosine  localized  head  border  hump-  p h o s p h a t a s e a c t i v i t y c a n be s e e n i n  a c r o s o m e and p o s t a c r o s o m a l  Figure  activity i s  acrosome and p o s t a c r o s o m a l  capitis,  distributed  Figure  activity.  The c o n n e c t e d  on t h e a n t e r i o r  5-nucleotidase a c t i v i t y  confined Figure  phosphatase  and t a i l .  Strong a l k a l i n e  galea  Figure  phosphatase  stained.  the  in  acid  segment  capitis,  also  Figure  strong  i n t h e head  equatorial  Figure  f o ralkaline  localization  galea  i n epididymal  1,640X.  Figure  like  localization  postacrosomal  i s localized  sheath,  and  tail.  i n the  Figure  33.  Malate dehydrogenase  strongly weakly Figure  34.  i n the middle p i e c e  Strong  dehydrogenase  to the middle  Lactate  middle  piece.  36.  37.  the  middle  and  activity  is  piece.  isocitrate  i n the middle  Strong  localized  dehydrogenase a c t i v i t y can beseen i n the  V e r y weak  localized Figure  succinate  is  head b a s e  piece.  35.  Figure  and  i n the p r i n c i p a l  confined Figure  activity  dehydrogenase  activity is  piece.  NADH d i a p h o r a s e a c t i v i t y i s c o n f i n e d t o  piece.  63 E.  DISCUSSION  1.  Phosphatases  Very the  strong  acid  phosphatase  postacrosomal sheath, t a i l  torial  region  tion.  Although several  phosphatase  (Wislocki,  Teichman  1970),  Stallcup,  1966;  and H a r t r e e , and  1969,  some i n v e s t i g a t o r s phosphatase  and have  mouse s p e r m a t o z o a  The  difference  e_t _ a l . , 1973 ),  incubation of  (Mathur,  the  and  Bernstein,  same s u b s t r a t e  and  acid  and  Guraya  phosphatase  demonstrated.  and  1971). a method  activity  on  and  ram,  acid B u c k l e y , 1969)  and  t o an and  In t h e p r e s e n t  mouse  1973),  association  Hartree,  1970;  s t u d y , however,  to that  applied  and  Bernstein  (1971),  (1975)  were u s e d  and  spermatozoa  was  i n mink  1973).  substrates  Unnithan,  due  boar,  1970),  the d i f f e r e n t  (Allison  similar  Allison  Unnithan,  for rabbit  o r t o be  and  (Teichman  o r no  and  ( B r y a n and  membrane  Sidhu  1971;  (Stambaugh  dependent  Melampy e t a l . ( 1 9 5 2 ) , T e i c h m a n (1971),  Allison  and H a r t r e e ,  Bryan  forma-  the  rabbit  little  obtained  fixations  t h e enzyme w i t h s t a b l e  Teichman  very  1971;  t i m e s ( P o i r i e r , 1975)  equa-  R o u s s e l and  1969,  (Allison  reported  t o be  1969;  mouse ( P o i r i e r , 1975 ),  rodent  in  o f t h e human  S i d h u , 1975),  i n the r e s u l t s  appears  i n the  reported  1952;  Bernstein,  i n the r a b b i t  and  (Chayen  Bernstein,  and  1971),  activity  spermatozoa  and  Guraya  p i g , hamster  have  i n spermatozoa  and  localized  a connected hump-like  (Melampy e t a l . ,  Teichman  1970;  Bernstein,  guinea  bull  was  especially  investigators  presence of a c i d  Hartree,  and  where i t p r o d u c e d  1950;  activity  a  by  Mathur  strong  64 The like  acid  phosphatase  structure  i n t h e head  resembles  t h e complex  (Teichman  and  phosphatase structural  in  this  Bernstein,  modification ( F i g . 2).  region  lysosomic  contact  a major  component  phatase  male a c c e s s o r y activity  was  spermatozoa et  i n both  o f .the human  activity  slight  the caput epididymis  t h e c o r p u s and  could Glover  not  also  enzyme.  acid  an  ovum.  phosphatase  f r a c t i o n a t i o n of r a b b i t  The  1950)  acid  washed  phosphatase  activity  i n the e x t r a c t  spermatozoa.  present  amounts i n  of seminal  Smith  Recently,  also  Summerson  acid  and  They  larger  e x c e p t ATP.  Stambaugh  that  i n the  enzyme was  free  the  (Melampy  shown  of the b u l l .  and  from  phosphatase  times higher  MacLeod  phos-  and  have  group  that  phosphatase,  derived  and b u l l  (1966)  three  epididymis.  However,  Acid  the  semen owes i t s p o w e r f u l  with increasingly  showed  activity  account f o r the  amounts o f t h i s  the phosphate  o f mink  i s a l y s o s o m a l enzyme.  Staccup  human s p e r m a t o z o a  split  (1973)  cellular trace  that  cauda  region  the s e m i n a l plasma  was  acid  to the  phosphatase  and  than i n spermatozoa  that  of  a c r o s o m a l enzyme d u r i n g  (Wislocki,,  R o u s s e l and  plasma  reported  related  might  of the acrosome,  located  demonstrated in  spermatozoa  pattern  i n t h e head  acid  which  spermatozoa  m a i n l y t o the s e m i n a l plasma  phosphatase  seminal  This  organs of r e p r o d u c t i o n .  a l . , 1952 ).  acid  (swellings)  suggested that  activity  spermatozoa  for rabbit  1971).  of a spermatozoon  (1964)  o f mink  a p p e a r s t o be  of r e l e a s i n g  initial  Mann  1969,  A strong  o f mink  action  region  illustrated  localization  spermatozoa  complex was f o u n d a t t h e d i s t i n c t hump-  Jones i s an (1973) of  (1946) plasma and  extrareported  subcellular  Poirier  (1975)  65 ultrastructurally between  t h e plasmalemma  t h e head  of mice  epididymis. rabbit and  demonstrated  and  subject  acid  and  Bernstein  phosphatase  a strong  from the  cauda  (1969) s u g g e s t e d  appeared  activity  that  t o be membrane bound  distribution.  t h e membrane o f mink s p e r m a t o z o a  activity  a c r o s o m a l membrane o f  collected  to s p e c i e s - s p e c i f i c  study demonstrated  phosphatase  the o u t e r  spermatozoa  Teichman bull  and  acid  of a c i d  collected  The  present  phosphatase i n  from  the  cauda  epididymis.  Acid sheath  phosphatase  and  localized  tail  activity  o f mink  i n human and  spermatozoa bull  Melampy e_t aJL. , 1 9 5 2 ) . involved also  not only  i n the acrosome, p o s t a c r o s o m a l  spermatozoa  This  i n the  lytic  action  s e m i n a l plasma  (1964).  Changes  spermatozoan migration  spermatozoa  and  1968;  Dingle,  spermatozoa  phosphatase i s  droplet  Glover,  o f t h e enzyme  which  1972;  t o show t h e i n v o l v e m e n t o f a c i d  groups  as d e m o n s t r a t e d by Mann i n mice  contains  and  the  acid  pass through the e p i d i d y m i s  Moniern and  but  of phosphate  m a t u r a t i o n ( P o i r i e r , 1975 ),  of the c y t o p l a s m i c as  acid  that  1950;  of the acrosome,  transport  i n the d i s t r i b u t i o n  heads d u r i n g  phosphatase  appear  and  to  (Wislocki,  suggests that  i n t h e d e p h o s p h o r y l a t i o n and  between  appears s i m i l a r  Bedford,  (Dott  1973)  phosphatase i n  spermatozoan m a t u r a t i o n . The  acid  correlation Haq  phosphatase  w i t h spermatozoan  and M u l l e n , 1949;  percentage  activity  of motile  of b u l l  concentration  R o u s s e l and spermatozoa  Stallcup, (Reid  semen has (Reid 1966),  a  positive  e_t a_l. ,  1948;  and w i t h t h e  et. al_. , 1948;  Roussel  66 and  Stallcup,  percentage 1966). the  of acid  isozymes  activity  rams  acid  phosphatase  phosphatase Such  inhibition  demonstrating of  (Chapter  and  t o show  acid  variability: of b u l l s  1973).  and  Back  three  and  five  L-tartrate  1973)  and  mink  i n that  inhibits  ( S i v a r a m and  in ejaculated  activity  Glover,  phosphatase i n human  Guraya  and  1952).  Bami, in  spermatozoa  in this  1972)  phosphatase  was  (Moniem and  Glover,  Alkaline  activity  found  study  was  and hand,  (Bavdek  and  and  i n the head bulls  rams  Glover,  activity  i n the r a b b i t  found  1950),  strongly  postacrosomal sheath  t h e enzyme  droplet  (Wislocki,  the o t h e r  distributed  investigators  S i d h u , 1975) On  was  acrosome,  to the c y t o p l a s m i c  spermatozoa  Fraser,  and  semen  be a r e a s o n f o r f a i l u r e s  (Gordon,  Although several  restricted  1952;  might  phosphatase  Moniem and  alkaline  in bull  the b u l l ,  i n human semen  i n the galea c a p i t i s ,  tail.  1970;  human  increases  I I I , Table 5).  Alkaline evenly  from  phenotype  t h e enzyme r e a c t i o n  the r a b b i t ,  Stallcup,  rabbit.  Yossifov,  activity  the  (1965) f a i l e d  between f e r t i l i t y  i n the s e m i n a l plasma  ( G e o r g i e v and  1971).  has  and  phosphatases  i n spermatozoa  with  and m i n e r a l s  However, S t a l l c u p  correlation  appear  (Roussel  vitamins  alkaline  a l . (1975) i n t h e r a t and Acid  correlation  spermatozoa  and  et. a_l. , 1 9 4 8 ) .  phosphatase  of  a negative  F e e d i n g of a d d i t i o n a l  any c o n v i n c i n g  et  but  o f abnormal  activity  (Reid  1966)  and  ram,  and  tail  (Melampy  e_t a l . ,  (Friedlaender  the r e a c t i o n  of  of  and  alkaline  not d e t e c t e d i n spermatozoa from the hamster, r a t 1972 ), and mouse  phosphatase  activity  (Mathur, of b u l l  1971).  spermatozoa i s  67 positively  correlated  spermatozoa activity  and  High  of  alkaline  correlated Alkaline and  phosphate  involvement  to t h i s , of  this  f o r e be  i n v o l v e d i n the e a r l y  The  (Moniem and  activity  the middle  (1975) showed middle  indicated been  plasma  Bavdek  1964)  the  i s very  little  of  utili-  of  alkaline  spermatozoa i s there-  epididymal  t o be  confined  G u r a y a and  i n the  Sidhu  postacrosomal  spermatozoa.  Mathur  sheath  (1971)  spermatozoa of mice.  presence  from  1949;  (1970)  activity  found  of  this  enzyme  spermatozoa  However, i n t h e  s p e r m a t o z o a were c o l l e c t e d  (Bern,  t h e enzyme m i g h t  c o u l d contaminate  result.  1965).  Glover  of r a b b i t  spermatozoa.  i n epididymal  that  also  1972).  t h e enzyme a c t i v i t y  positive  and  1966).  dephosphorylation  and  The  maturation  Glover,  p i e c e o f mink  suggested  semen i s  the  the  Stallcup,  (Stallcup,  o f 5 - n u c l e o t i d a s e was  i t s absence  and  the  with  enzyme i n t h e g l y c o g e n  transit  p i e c e of b u f f a l o  (Mann,  a false  there  cent  i n the c y t o p l a s m i c d r o p l e t  spermatozoa  while  g r o u p s a c r o s s membranes  during epididymal  has  per  i s associated with  reduced  and  in bull  w i t h i n the c y t o p l a s m i c d r o p l e t .  phosphatase  to  spermatozoa,  (Roussel  activity  In a d d i t i o n  an  live  spermatozoa  high nonreturn  phosphatase  Mann, 1 9 6 4 ) .  of  of m o t i l e  i s negatively correlated  phosphatase  t r a n s p o r t of  zation  plasma  abnormal  with  suggested  the p e r c e n t a g e  the p e r c e n t a g e  i n seminal  percentage  with  present  and  due  seminal produce  study,  the cauda e p i d i d y m i s  chance of contamination  in  to  It  mink  where seminal  plasma. Mann seminal adenylic  (1964) summarized  plasma acid,  the r o l e  as d e p h o s p h o r y l y z i n g and  other  of  5-nucleotidase  i n the  ribose-5-phosphate,  nucleotides.  G u r a y a and  Sidhu  (1975)  68 assumed  i t s role  Moreover, sheath  in permeability  lysosomal  action  5-nucleotidase  acrosome The  to  and  this  rabbit  and  acrosomal  et. _ a l . , 1975 ) s u g g e s t s i t s  spermatozoa enzyme  activity  the m i d d l e p i e c e could  Multamaki  might  activity  o f mink  not l o c a l i z e  human, M a t h u r  (1971)  found  Glucose-6-phosphate  plays  an e s s e n t i a l  because  Consequently,  of  raises  the s t r o n g the q u e s t i o n  and  appeared  and/or  i n the  i n g l y c o g e n metai n glycogenesis,  Fisher,  hydrolyses  1962). glucoses-  of glucose-6-phosphate  (1964)  (Anderson  stated  that  and  and the  seminal  plasma  activity  o f G-6-Pase  i n mink  whether  g l u c o s e and  glycogen  semen i s r e l a t i v e l y  high  compared  mammals.  Although rabbit  which  o f the  spermatozoa.  role  the d i s p o s t i t i o n  A l t h o u g h Mann  of spermatozoa  other  o f mice  of glycogen metabolism  low,  Gordon  i t s activity  glucose c o n t e n t i n spermatozoa  mammals was  content  found i n  Although  ( K r e b s and  G-6-Pase a c t i v i t y ,  1970). and  tail  glycolysis  influences  spermatozoa  to  and  the r e g u l a t i o n  glycogen  such  confined  i t i s a common i n t e r m e d i a t e  glycogenolysis,  Personne,  n o t been  t h e enzyme i n s p e r m a t o z o a  and  and  has  spermatozoa.  base,  phosphate,  not d i s p l a y  o f G-6-Pase was  t i p , head  bolism  However,  or i n the p o s t a c r o s o m a l s h e a t h .  strong  (1973)  i n the p o s t a c r o s o m a l  involvement' i n f e r t i l i z a t i o n .  i n mink  because  process.  S i d h u , 1 9 7 5 ) , a c r o s o m e and c y t o p l a s m i c d r o p l e t  ( G a r b e r s e_t a J . , 1970;  the  transport  t h e p r e s e n c e o f enzyme a c t i v i t y  ( G u r a y a and  function  and  ADPase a c t i v i t y  human s p e r m a t o z o a  t o be  distributed  was  not found i n  (Gordon,  strongly  1973),  this  i n the t a i l  enzyme  and  weakly  in  69 the  acrosome  With r e g a r d  and  t o ATPase,  galea c a p i t i s , activity  confined  with  .the head  previous  1967,  i n rams;  1972,  1971,  localized 1958;  Baccetti tubules noted  Gordon  and  i n mice).  and  and  activity Hilmoe,  Quinn  Quinn  and  filament  Rowe, 1965;  i s also 1953 ) and  with  around  on  Gibbons  v/as  Gordon  1967,  and  Barrnett,  i n guinea ATPase  fibers  pigs; activity  (Nelson, and  1958;  central  i t i s t o be  doublets  o f the 1966;  F r o n k , 1972 ).  (Mann, 1945a,  is  (Nelson,  ( G i b b o n s , 1965,  droplets  Guraya  i n boars; Durr  Particularly,  and  1954,  V o g l m a y r e_t  of the t a i l  the o u t e r  activity  1968,  1968,  the doublets  i n semen  in cytoplasmic  activity  lowest  1966,  strong is in  (Nelson,  White,  complex  o f ATPase  present  i n the  result  and W h i t e ,  1967), dense  located  axoneme, a r e c o n s t i t u t e d Gibbons  1957,  e t a_l. , 1 9 7 3 a ) .  t h e arms,  ATPase  Ultrastructurally,  Barrnett,  (Baccetti  This  spermatozoa  Barrnett,  e t al_. , 1975 ). and  that  to the t a i l  U e s u g i and Yamazoe,  i n the a x i a l  Gordon  i n that  i n human; N e l s o n , 1958,  in rats;  Mathur,  in bulls;  v/as d e t e c t e d  spermatozoa.  reports  Burnasheva,  S i d h u , 1975,  al.,  o f mink  spermatozoa.  p o s t a c r o s o m a l s h e a t h and  o f o t h e r mammalian and  a l . , 1969, et  and  almost e x c l u s i v e l y  Engelgardt and  a weak a c t i v i t y  acrosome,  i n the t a i l  agreement  in  p o s t a c r o s o m a l s h e a t h o f mink  ATPase  Heppel  and  ( G a r b e r s e_t a l . ,  1970). ATPase adenosine phate with  and ADPase b r i n g  d i p h o s p h a t e and the r e l e a s e  (Mann, 1 9 6 4 ) . between  The  the h y d r o l y s i s  successively  transport  of a l k a l i  seminal plasma  o f ATP  to  t o a d e n o s i n e monophos-  of energy f o r spermatozoan  active  s p e r m a t o z o a and  about  motility  metal  involves  a  ions  sodium-  70 potassium  a c t i v a t e d ATPase  and W h i t e , the  1967b,  1968).  Gordon  i n v o l v e m e n t of ATPase  differentiated  ATPase  (Uesugi  activities  of f i x a t i o n ,  specificity,  and  sensitivity  1966;  and  Barrnett,  1973).  Therefore,  these ATPases, constitute roles  2.  two  i n spermatozoan  and  located  and  to i n h i b i t o r s  1967; and  activity  Voglmayr  Barrnett  distinct  pH  substrate  (Uesugi  and  Yarnazoe,  e t a l . , 1969;  (1967)  and  on  are  Gordon,  suggested that  morphologically  u n i t s , might  play  separate  motility.  Oxidase  esterase  e_t a_l. (1971)  tail  (1967) s u g g e s t e d  a c t i v a t i o n ions,  activity  i n t h e b a s e o f t h e head  and M e i z e l  Barrnett  Quinn  i n spermatozoa based  which c y t o c h e m i c a l l y  Non-specific  head  Gordon  at least  Esterase  and  Yarnazoe, 1966;  i n spermatozoan c a p a c i t a t i o n . These  optimum, e f f e c t s  Gordon  and  of b u l l  and  reported  spermatozoa  i n the middle p i e c e  i n mink  spermatozoa  middle piece.  esterase and  of mice  Meizel  activity  Mathur  was (1970)  i n the  (1971) 'showed i t s  s p e r m a t o z o a . On t h e o t h e r  hand, e s t e r a s e a c t i v i t i e s were f o u n d t o be c o n f i n e d o n l y i n t h e a c r o some o f b u l l s p e r m a t o z o a ( B r y a n and U n n i t h a n , 1972) and m i c e and h a m s t e r s p e r m a t o z o a ( B r y a n and U n n i t h a n , 1 9 7 3 ) . N i e m i and Kormano (1965) , h o w e v e r , c o u l d n o t d e t e c t t h e e s t e r a s e a c t i v i t y i n t h e a c r o s o m e of the  r a t spermatozoa.  The  esterase  s e m i n a l plasma of the b u l l  1971)  and  droplet phoretic certain  rabbit  (Bradford  study, Meizel esterase  (Meizel,  e t al.,  ( M e i z e l e t a l . , 1971)  reaction also occurred Meizel  1976a) and  of the b u l l .  e t a _ l . (1971)  activity  1970;  the  By  an  demonstrated  o f s p e r m a t o z o a was  due  in  et a l . ,  cytoplasmic electro-' that  a  to absorbed  71 seminal  plasma  Recently esterolytic cellular  Bradford  activity  cumulus  ester  layer.  be c l a s s i f i e d  Kjessler, The  and  dispersed  and s e m i n a l p l a s m a  that  strains.  (Meizel,  a difference  (Beckman and  spread i nthe  o f mink  i n the t a i l  spermatozoa.  o f spermatozoa  of this  enzyme h a s a l s o b e e n 1972),  bulls  1971),  g o a t s , rams  (Mukherjee,  (Mathur  1974),  and r a b b i t s  (Beatty,  1970), the  t h e DOPA o x i d a s e i s p r e s e n t  t h e DOPA a c t i v i t y  enzyme  shows s t r o n g The of  (Beatty,  DOPA o x i d a s e r e a c t i o n ,  spermatozoa,  rabbit  (Beatty,  Mukherjee a  DOPA r e a c t i o n  because  (1964)  definite  activities.  i s associated  that  correlation  1964),  washed  mice  1956).  i s not dependent spermatozoa  (Beatty, on also  1956). i s the melanizing  activity  with somatic pigmentation of the  1956) and human ( A c k e r m a n , and P a n t  found i n  i n seminal plasma  o f spermatozoa  i n seminal plasma  between  ( M u k h e r j e e , 1964;  P a n t and M u k h e r j e e ,  Although  demonstrated  i n the degree of a c t i v i t y  o f human ( A c k e r m a n ,  and B e a t t y ,  esterase  1970).  was w e a k l y  showed i t s a c t i v i t y  The a c t i v i t y  spermatozoa  contained  dispersed the  the reported  o f t h e human  p o s t a c r o s o m a l s h e a t h and t a i l  he r e p o r t e d  which  i s o z y m e s o f e s t e r a s e were  DOPA o x i d a s e a c t i v i t y  (1971)  the i n t r a -  while hyaluronidase  proposed  that the  as EC 3.1.1 and p r o b a b l y a s EC 3.1.1.2,  1968) and b u l l  acrosome,  reported  of the corona r a d i a t a  also  Several  spermatozoa  Mathur  o f t h e acrosome  linkage,  They  arylesterases. in  e t al_. (1976a,b)  cement m a t e r i a l  an e s s e n t i a l  might  esterases.  and M u k h e r j e e  between  Afterwards, Beatty  coat  (1971)  colours  (1970)  1972).  also  However,  could  not f i n d  and m e l a n i z i n g admitted that the  72 melanizing related.  activity On  and  the other  pelt hand,  spermatozoa  i s negatively  temperature  shock,  nigrosin) ram  The  DOPA a c t i v i t y  colour  spermatozoa  reported and  the d i f f e r e n c e  by  either  activity  and w e a k l y  1964;  (unstained  rates Pant  in eosin-  of the b u l l ,  with the degree of the  activity  between  o r by  1971).  natural  Although Mukherjee  i n the m e l a n i z i n g  or s t r a i n ,  goat,  and M u k h e r j e e ,  t h e same a n i m a l , M a t h u r  confined (Balogh cat,  (1964) animals  and  Beatty  activity  both.  which  dog  1964;  ( B a l o g h and  and  small  i s in close MDH  trophoretically (Goldberg,  stimulated  by  The egg  1971), 1964).  base  the. mouse  human, r a t , g u i n e a p i g ,  o f t h e enzyme m o s t l y i n in bull  of which  spermatozoa  of t h i s  of malate  Human s p e r m a t o z o a  but  o f mink  o f t h e enzyme i s  from  with the r e s u l t  MDH  piece  However, M o h r i e t a l .  o f the head  activity  yolk  head  reaction  i n the o x i d a t i o n  cycle.  i n the middle  of spermatozoa  Cohen,  portion  distinct  1963).  strong  Mathur,  agreement  acid  and  the d i s t r i b u t i o n  i s involved  tricarboxylic  strongly  piece,  to the middle p i e c e  demonstrated  tail  appeared  A comparatively  and C o h e n ,  and  (1965)  o f MDH  i n the p r i n c i p a l  spermatozoa.  is  to  Dehydrogenases The  the  with resistance  of l i v e  1970).  a variation age  directly of  of melanizing from  n o t be  activity  fructolysis  (Beatty,  (1974) d e m o n s t r a t e d  3.  and  i s correlated  between e j a c u l a t e s  affected  correlated  (Mukherjee,  o f t h e semen  could  the m e l a n i z i n g  the p r o p o r t i o n  spermatozoa,  and  colour  MDH^  o f MDH  inhibited  i n the  contain  has more  i n bovine  study.  two  elec-  activity  spermatozoa  by c h o l e s t e r o l  (Smith  73 et  al.,  1956 ) .  The to  activity  o f SDH  the middle p i e c e  with  the f i n d i n g s  from  t h e human  (Hrudka,  of other  rabbit  1965),  mouse  guinea  p i g , c a t , and  Nelson  (1955)  region  reported  SDH  complement  1964;  and  t h e SDH  enzyme  i n spider  This  is in  Hrudka,  Cohen,  Cohen,  agreement  1965), 1963;  Hrudka,  However,  in bull  and m i d d l e  s u b s e q u e n t l y (1959)  i n the n i n e outer  sperma-  piece demonstrated  dense  fibers  flagellum.  Furthermore, there  spermatozoa  which  Kamboj,  bull  1964), r a t ,  1964).  activity  confined  spermatozoa  Valentine,  ( B a l o g h and  He  s t u d y t o be  studied  B a l o g h and  of 2 : 1 .  ( G u p t a and  who  i n the p r i n c i p a l  the r a t spermatozoa activity  Cohen,  that  concentrated  the presence of t h i s of  workers  1971; dog  in this  spermatozoa.  (Edwards  (Mathur,  in a ratio  found  o f mink  ( B a l o g h and  1965),  t o z o a was  was  lack  i s no  mitochondrial  1962).  At t h e i n i t i a l s t a g e i n t h e e l e c t r o n t r a n s p o r t s y s t e m , SDH p l a y s a k e y r o l e i n sperm m e t a b o l i s m ( N e l s o n , 1959 ). can u t i l i z e energy  the t r i c a r b o x y l i c  (Bishop,  1962)  and  (Lardy  spermatozoa  cannot u t i l i z e  because  the spermatozoa  Johnsen Nelson  (1954,  malate cell  1955)  were c o n c e n t r a t e d  fibers  suggested  (Nelson,  oxygen  and  acid  Phillips,  cycle  Phillips, that  1945).  b o t h SDH  and  that  ATPase  acids,  1945) to these  However, ATPase  flagellar was  of  bull  (Koefoed-  i n the spermatozoan 1959)  uptake  dicarboxylic  membrane i s i m p e r m e a b l e  demonstrated  activities and  (Lardy  L a r d y and  source of  However, e j a c u l a t e d  the f o u r - c a r b o n  of the t r i c a r b o x y l i c  and Mann, 1954;  as a m a j o r  stimulates  e_t _ a l . , 1 9 4 5 a , b ) .  f u m a r a t e , and  intermediates  cycle  succinate  spermatozoa  succinate,  acid  Mammalian s p e r m a t o z o a  74 synthesized supply  within  o f ATP  spermatozoa importance by  metabolism  yolk  and  and  drying  between r e d u c t i o n  fails  spermatozoa (Keilin  fully  yolk  time,  ( S m i t h e t al_. , 1 9 5 6 ) .  withstands freezing 1949)  SDH  The  While  SDH  activity,  activity  activity  (-196°C) and and  the  motility,  stimulate  or i n h i b i t  in  has b e e n c o n f i r m e d  (Smith e t a l . , 1956).  to stimulate  and H a r t r e e ,  o f SDH  elucidated,  to spermatozoa  c a r o t e n e o f egg  spermatozoa  assured a continuous  Although the r o l e  n o t been  nonreturn rate  lecithin  bovine  has  of dehydrogenases  cholesterol  and  for i t s motility.  the r e l a t i o n s h i p s  longevity  in  the f l a g e l l u m  washing  of  of  SDH  subsequent  ( J o n e s and  Holt,  1974). The in  LDH  activity  the m i d d l e p i e c e .  (Balogh  and  Cohen,  1964; C h u r g e t  Storey  and  and  Personne,  a l . , 1974),  rabbit  -(Churg e t a l . ,  r a t ( B a l o g h and  g u i n e a p i g , c a t , and  matrix  o f m i t o c h o n d r i a ( A n d e r s o n and  activity  LDH  activity  M o h r i e_t a _ l . (1965) located  i n the p r i n c i p a l  middle piece  o r head  region  strain,  i n mice  o r by b o t h  Most are  mammalian  composed  addition  spermatozoa (Mathur, tissues  o f H and M  1971;  to these isozymes,  localized  and  i n the the  t h a t most o f t h e  LDH  1970).  rather  than i n the The  t o age  and B e a t t y , LDH  (Wilkinson,  testes  Anderson  ( B a l o g h and  according  five  1974;  On  piece  Mathur  contain  1964;  spermatozoa.  differs  subunits  dog  Personne,  reported  of b u l l  ( B a l o g h and  Cohen,  i s primarily  was  activity  i n t h e mouse  human  1964).  hand,  localized  1971),  Cohen,  other  The  i s weakly  Mathur,  1975),  1970),  spermatozoa  T h i s agrees with those found  1964;  Cohen,  Kayne,  o f mink  isozymes 1965).  spermatozoa  LDH or 1974). which In  contain  75 a  large  1963; is  amount o f a s i x t h  Goldberg,  and  Moran,  plasma  reduces  1975b).  second low  spermatozoan  cancer  rabbits  (Eliasson,  The  The m i d d l e  f o r aerobic  subunit  which  (Stambaugh The oxygen  In be  blockage (Goldberg,  (between t h e f a c t o r of  tissue  The  as i n p r o s t a t e  isozymes  piece,  site  are d i f f e r e n t l y of aerobic  t h e X and H s u b u n i t s  suited  The t a i l  fibers  glycolysis in which  are best  contain  the M  f o r anaerobic glycolysis and B u c k l e y ,  t h e b a l a n c e o f NADH ~NAD  u n d e r low  2  i n the spermatozoan  metabolism  1969).  ( A n d e r s o n and  1970).  spite  identified  spermatozoa. who r e p o r t e d piece  LDH band  1968).  signifi-  to the m o t i l i t y  a n d P o s t , 1967; Stambaugh  LDH r e g u l a t e  Personne,  w i t h LDH-X  ( P r a s a d et a_l. , 1976 ) .  glycolysis.  i s best  tension  (Eliasson,  1968).  the m i t o c h o n d r i a , c o n t a i n s suited  density  i n malignant  p a r e n t form o f f i v e  distributed.  (Gregoire  o f t h e embryo m o r t a l i t y  LDH b a n d s ) r e l a t e s  o f LDH,- i n c r e a s e s  from  o f LDH-X i n t h e s e m i n a l  and m i c e  an e x t r a  semen  LDH-X  of these animals through  and i n c r e a s e  count  1969).  h a v e no LDH-X  with the spermatozoa  Furthermore,  and t h i r d  activity  Semen s a m p l e s  the f e r t i l i t y  fertilization  1973,  isozyme.  o f female  ( B l a n c o and Zinkham,  S c h a t z and S e g a l ,  1972, 1973) a n d t h e a c t i v i t y  correlates  cantly  LDH-X  and v a s e c t o m i z e d males  Immunization  of  1963, 1975a;  a sperm-specific  oligospermic  isozyme,  o f mice  o f r e p e a t e d a t t e m p t s , no r e a c t i o n  product  t o show t h e p r e s e n c e o f PDH a c t i v i t y This that  result this  differs  from  that  enzyme was l o c a l i z e d  spermatozoa.  could  i n mink  o f Mathur  (1971)  i n the middle  As PDH i s i n v o l v e d  i n t h e hexose  76 monophosphate not  utilized  and W a l t o n that  shunt pathway,  by e p i d i d y m a l s p e r m a t o z o a  (1966b)  and V o g l m a y r  t h e r e was no h e x o s e  testicular The  I t appears  middle piece  l i z e d IDH a c t i v i t y .  e_t a _ l . (1970)  spermatozoa  o f mink  this  pathway i s  i n the mink.  monophosphate  or ejaculated  that  Bishop  also concluded  shunt a c t i v i t y  in either  of b u l l s .  spermatozoa  showed  faintly  loca-  I t i s consonant with the r e s u l t s i n spermatozoa  f r o m t h e mouse ( B a l o g h and C o h e n , 1964; M a t h u r , 1 9 7 1 ) , rat., g u i n e a pig,  c a t , and dog ( B a l o g h and C o h e n ,  involved acid  i n the oxidation  cycle.  confined  and  In a l l t e s t e d  o f MDH  principal  reaction related  for  motility  their  Strong  the r e s u l t  (NAD)  action  and Masek,  dehydrogenases  on coenzyme  from  head  base  Dehydrogenase  t o make u s e o f  substrates  was c o n f i n e d  This  closely  to the  agrees with  1964; M a t h u r ,  1961; B a l o g h and C o h e n ,  ( B a l o g h and C o h e n , involved  acid  cycle  nicotinamide  f o r m i n g r e d u c e d NAD  hydrogen  reaction).  ( B a l o g h and C o h e n ,  pathway and t r i c a r b o x y l i c tive  with the  1965).  spermatozoa.  c a t , d o g , and human Several  was  m e t a b o l i c p r o c e s s e s as a s o u r c e o f e n e r g y  i n t h e mouse  guinea p i g (Birns  activity  sheath of the middle piece i s  of spermatozoa  (Hrudka,  o f mink  spermatozoa  (negative  NADH d i a p h o r a s e a c t i v i t y  piece  i n the t r i c a r b o x y l i c  i n t h e m i d d l e piece,,  i n the mitochondrial to the a b i l i t y  The IDH i s  dehydrogenase  o f mink  p i e c e ) and PGD  and a e r o b i c  rat,  samples,  (distributed  anaerobic  middle  of i s o c i t r a t e  to the middle piece  exception  1964).  1971),  1964),  1964).  i n t h e Embden-Meyerhof depend  for their  adenine  oxida-  dinucleotide  (NADH) t h r o u g h t h e t r a n s f e r o f t o t h e coenzyme.  The r e d u c e d  77 coenzyme  (NADH) i s o x i d i z e d back  t o NAD by NADH  ( C h a y e n e_t _a_l. , 1 9 7 3 ) .  Therefore,  role  and r e s p i r a t o r y  i n both Although  glycolytic  (with  the exception  variation study acid  t h a t mink  plays  metabolism.  i n the spermatozoa  i n mink e p i d i d y m a l  PDH was n o t l o c a l i z e d )  of l o c a l i z a t i o n . spermatozoa  a key  spermatozoa  there  The r e s u l t s  utilize  of other  i s some of this  the t r i c a r b o x y l i c  c y c l e a n d Embden-Meyerhof pathway f o r v a r i o u s o x i d a t i v e ,  reductive of  that  i n the sites  suggest  NADH d i a p h o r a s e  t h e enzymes r e p o r t e d  mammals h a v e a l s o been d e t e c t e d  diaphorase  and o t h e r  mink s p e r m a t o z o a  coitus  (Enders,  enzyme  activities.  biochemical  processes.  i n the uterus  1952) m i g h t  The l o n g  f o r more t h a n  be a c c o u n t e d  survival  19 d a y s  post-  f o r by t h e s t r o n g  78 F.  SUMMARY Epididyrnal  cellular  spermatozoa  localization  Very  of several  strong acid  equatorial  r e g i o n o f t h e head  galea c a p i t i s ,  of  while  acrosome,  connected  the e q u a t o r i a l  activity  post-acrosomal  sheath  reaction  and  phosphatase  sheath.  a structural Strong  The a c t i v i t y  was v e r y s t r o n g . strongly  was w e a k l y  localized with  was f o u n d  Deeply  alkaline  weak w h i l e  phos-  acrosome,  piece.  that of  The a c t i v i t y  o f ADPase  and i t was  by A T P a s e .  i n t h e acrosome,  border  of 5-nucleotidase  i n the t a i l  stained  i n the  modification i n  i n t h e a c r o s o m e and p o s t a c r o s o m a l  galea c a p i t i s  tail,  and p r i n c i p a l  were c o n f i n e d t o t h e m i d d l e  A T P a s e was d i s t r i b u t e d weakly  i n the  on t h e a n t e r i o r  o f 5 - n u c l e o t i d a s e appeared  glucose-6-phosphatase  t a s e s were  prominences  and t a i l .  The  The  (middle  was d e t e c t e d i n t h e g a l e a c a p i t i s ,  glucose-6-phosphatase  localized  was f o u n d  strong a c t i v i t y  spermatozoa.  and  and  and t a i l  segment . s u g g e s t  t h e head r e g i o n o f mink phatase  activity  and p o s t a c r o s o m a l  hump-like  to study the  enzymes.  phosphatase  p i e c e s ) of spermatozoa  stained  o f mink were u s e d  sheath.  Most  postacrosomal  phospha-  sheath,  t h e e x c e p t i o n o f 5 - n u c l e o t i d a s e and g l u c o s e - 6 -  w h i c h were c o n f i n e d t o t h e m i d d l e  Weak n o n - s p e c i f i c b a s e o f t h e head  esterase activity  and m i d d l e  o x i d a s e was w e a k l y  spread  sheath,  Although  and t a i l .  piece.  v/as l o c a t e d  The a c t i v i t y  i n the acrosome, the a c t i v i t y  d e h y d r o g e n a s e was d i s t r i b u t e d  piece. i n the  o f DOPA  postacrosomal  o f malate  i n t h e b a s e o f t h e head  79 and  tail,  the rest  (succinate,  o f the dehydrogenase  lactate,  and i s o c i t r a t e  activity  dehydrogenases except  6 - p h o s p h o g l u c o n i c d e h y d r o g e n a s e ) was c o n f i n e d piece.  The a c t i v i t y  strongest  among  of succinate  t h e examined  NADH d i a p h o r a s e was s t r o n g l y  and  NADH d i a p h o r a s e ) were c o n f i n e d  the  activity  to  t h e head  phosphatase  enzymes  confined  metabolic  was t h e  The a c t i v i t y o f  to the middle  piece.  (glucose-6-phosphatase, dehydrogenases, to the middle piece,  o f malate dehydrogenase base  to the middle  dehydrogenase  dehydrogenases.  The  examined  and p r i n c i p a l  and e s t e r a s e ,  while  was f o u n d t o be e x t e n d e d  piece.  The l y t i c  were l o c a l i z e d  enzymes,  acid  i n the acrosomal  portion. Although localization, exception mink  t h e r e was some v a r i a t i o n a l l examined  enzymes  i n the s i t e of  i n this  study, with the  o f 6 - p h o s p h o g l u c o n i c d e h y d r o g e n a s e , were  spermatozoa-as  found i n spermatozoa of other  localized i n mammals.  80 Chapter I I I ULTRASTRUCTURAL AND CYTOCHEMICAL CHANGES OF MINK SPERMATOZOA COLLECTED FROM THE REPRODUCTIVE TRACT AND SEMEN  A.  INTRODUCTION Spermatozoa a r e produced  the  testis  and u n d e r g o  where t h e y  physiological fertilizing in  changes o c c u r  ability.  the t e s t i s  with  through  logical  maturity,  per cent  to c e r t a i n  contact  metabolic  they  and  to acquire take  place  i n the e p i d i d y m i s . changes  take  enter  the e p i d i d y m i s .  place  During  spermatozoa a t t a i n  physio-  as m e a s u r e d by t h e f e r t i l i z a t i o n spermatozoa.  temperature  i n negative  Variations i n  shock,  surface charge,  chemical  composition,  p a t t e r n , and s p e c i f i c  r a t e and  alkalinity  or a c i d i t y ,  agglutinating property, enzyme  gravity  response  distribution,  have b e e n  noted not  o n l y b e t w e e n e p i d i d y r n a l and e j a c u l a t e d s p e r m a t o z o a between  During  chemical  changes  physiological  the e p i d i d y m i s ,  of motile  ability,  alterations  place.  Mammalian s p e r m a t o z o a a r e n o t p h y s i o -  staining,  differences  i n spermatozoa  hand, most  m a t u r e when  passage  the  takes  Most m o r p h o l o g i c a l  minor  the e p i d i d y m i s .  logically  ejaculation  tubules of  i n the epididymis  a number o f m o r p h o l o g i c a l ,  On t h e o t h e r in  f u r t h e r maturation  are stored u n t i l  spermatogenesis,  i n the seminiferous  spermatozoa c o l l e c t e d  from  the proximal  but a l s o  and  distal  segments o f t h e e p i d i d y m i s . The the  i n t r o d u c t i o n and i m p r o v e m e n t  development  of continuous  of e l e c t r o n  spermatozoan  microscopy,  collecting  techniques  from  the t e s t i s  and e p i d i d y m i s ,  and t h e i m p r o v e -  ment o f h i s t o c h e m i c a l m e t h o d s have f a c i l i t a t e d elucidation in  of maturation  s e v e r a l mammals.  spermatozoan therefore  mechanisms o f s p e r m a t o g e n i c  However,  maturation.  there  cells  i s no r e p o r t on mink  The aim o f t h e p r e s e n t  the i n v e s t i g a t i o n  spermatozoa.  the  of the maturation  study  changes  was i n mink  82 B.  REVIEW OF  1„  Early  RELATED  Studies  Aristotle reproduction  examination (Meyer,  taken von  1850)  LaVallete the  of  and  d e s c r i p t i o n of  cells  Regaud  technique  the  conducted  t e s t e s of  the  first by  animal  of  gonadal  microscopic  Ham  in  1677  spermatozoa this  the  tract.  v/as  interstitial  species.  spermatozoa  developed  discovery  1865).  the  a l l examined  reproductive  were m o r p h o l o g i c a l l y  (1876);  of  In  1841  from  cells  followed  by  c e l l , (Leydig,  The  various  classified  by  (1901) e s t a b l i s h e d t h e  germinal  von  concept  of  spermatogenic c y c l e . The  century  h i s t o l o g y of by  reported The  classic  and S e r t o l i ' s c e l l s ( S e r t o l i ,  epithelium  physiology  d i f f e r e n c e s between  that  testis,  Tract  Dumas ( 1 8 2 4 a , b ) r e p o r t e d  the  parts  the  interval,  i n the  discovered the  microscopic  the  P r l v o s t and  various  In  studied  s p e r m a t o z o a was  spermatozoa  Kolliker  residing  B.C.)  characterized  from  Reproductive  introducing  1938).  then  the  A f t e r a long of  presence of They  (300  by  extirpation.  of  LITERATURE  Becker  the  secretory  1897;  Aiger,  were r e p o r t e d 1924,  von  extended testis  to  the  (1857).  activity 1900) by  beyond  concentrates  Hammar  was  and von  of  the  vas the  the  the  epididymal  absorbing  Mollendorff that  the  dilute  Aiger  (1900)  epithelium  (Hammer,  of  the  Young  f u n c t i o n of  spermatozoan  19th  duct.  ability  the  epididymis  (1933).  the  spermatozoa  Furthermore,  i n the  epididymal  ( 1 9 2 0 ) and  t r a n s p o r t a t i o n of  deferens.  described  (1897) and  r e g i o n a l d i f f e r e n c e s i n the  Lanz emphasized  the  epididymis  epididymis from  the  epididymis  suspension,  In  retains  83 spermatozoa  for sufficient  s t o r e s matured spermatozoa tive  tract  fertility Finally,  spermatozoa  collected show an (Redenz,  Young  from  1926;  end  of  The  Cytoplasmic  along  i s noticed  the c y t o p l a s m i c d r o p l e t . droplet  droplet  of  piece.  spermatozoa  tract  from  1937;  Branton  1963a).  The  and  components  found The  and  they  reproducand  Yochem,  by  end  Redenz  1930).  spermatozoa  showing  compared  a  to  p i e c e and  at  two the  relative the  through and  of  repro-  position  finally  of the  sloughed o f f  The  cytoplasmic  1934;  Hancock,  1957a;  1961).  The  of  bounded  the d r o p l e t  middle  and  the d i s t a l  of  the  reaches  1948;  of  types  Bloom  distal  and  Bedford, is  droplet  vesicular  a distinct the  Selivanova,  droplet  proximal  t u b u l a r and by  end  the r e p r o d u c t i v e  (Lagerlof,  1947;  that  Hart,  the  p r o p o r t i o n s of these  level  from  the d r o p l e t  morphological  t h e neck r e g i o n o r d i s t a l  structure  different  obvious  (1924) r e p o r t e d t h a t  a r e removed  internal  (Rao  most  i n the p r e s e n c e  become s m a l l e r as piece  transit  the passage  Salisbury,  Nicander, of  the  mammalian  the cauda e p i d i d y m i s .  vary with  which  considerably (Bloom  reached  i s usually  the middle  that  The  moved down t h e m i d d l e  spermatozoa  of  1931;  i n the d i s t a l  Droplet.  i n spermatozoa tract  The  for motility  1929a,b,  and  the e p i d i d y m i s .  a.  as  Young,  during epididymal  M o r p h o l o g i c a l Changes  ductive  capacity  (1931) d e m o n s t r a t e d  2.  change  ejaculation.  successive levels  i n c r e a s e of f e r t i l i t y  proximal  to a l l o w maturation,  until  increasing  undergo m a t u r a t i o n fold  time  membrane end  Nicander,  of  the  1961;  84 Bedford,  1965b).  While  (1948) s u g g e s t e d piece, the  Kojima  droplet The  Hart,  1964)  1958),  accurate  droplet  in  that  still  that  index  fertilizing movement  prostate  (Selivanova,  investigators the droplet  was  a nutritive  Bedford  could  (1967b) a l s o could  relative  maturity  of spermatozoa  The A c r o s o m e .  Acrosomal  s p e r m a t o z o a were f i r s t reported  spermatozoa greater  than  from  that  light  an  suggested  that the  of spermatozoa.  that  t h e p r e s e n c e and  1963a).  i n maturing  bovine  by M u k h e r j e e and B h a t t a c h a r y a  epididymis  o f t h e head o f was  significantly  spermatozoa.  and e l e c t r o n m i c r o s c o p i c  shown s t r u c t u r a l m o d i f i c a t i o n s ,  not give  n o t be o f i m p o r t a n c e  (Bedford,  the dimension  of e j a c u l a t e d  (1967a)  are i n d i c a t i o n s of the  changes  observed  the caput  that  Subsequent  accepted  droplet  substance  i n terms o f  t h e d e v e l o p m e n t o f m o t i l i t y and m a t u r i t y i t i s generally  1948; Mann,  Orgebin-Crist  of spermatozoan maturation  of the cytoplasmic  (Rao and  the r o l e of the cyto-  of the d r o p l e t  and l o s s o f t h e d r o p l e t  (1949) who  1937; B i a l y  (Rao and H a r t ,  remains obscure.  location  gland  regression.  the absence  ability.  Nevertheless,  b.  vesicle  f o r spermatozoan m a t u r a t i o n ,  maintained  the r e s o r p t i o n of  and p h y s i o l o g i c a l s a l i n e s o l u t i o n  several  considered  plasmic  o f f at the middle  (1963) r e p o r t e d  seminal  1948) i n d u c e d r o p l e t  required  slid  o f the cauda e p i d i d y m i s ,  1937),  Although  ( 1 9 3 7 ) and Rao and H a r t  spermatozoa.  secretion  Smith,  the droplet  and I s h i k a w a  by  (Selivanova, and  that  Selivanova  including  studies  a reduction  have i n the  85 acrosomal guinea  d i m e n s i o n s .of s p e r m a t o z o a  p i g ( F a w c e t t and H o l l e n b e r g ,  Phillips, rabbit  1969a), c h i n c h i l l a  (Bedford,  1963a,  Koefoed-Johnsen,  and  N i c a n d e r , 1 9 7 1 ) , monkey (Dickey,  hare  and  significant structure  duct  size  fertilizing of  1975).  (Jones,  was  (Bedford  ability.  relationship  and  of the d r o p l e t .  Hollenberg  (1963)  fertilizing tion of  o f the acrosome  the head  spermatozoa. acrosomal  c.  suspected  capacity  1975).  on  However,  e_t a_l. , 1 9 7 3 ) , reduction  Membrane.  On  no  i n the only  head,  that  there  of  i n matured  i s perhaps  no  t h e acrosome m a t u r a t i o n hand,  Fawcett  and  the i n c r e a s e i n  affect  the r e s u l t  of  o f accompanying t h e swimming  the f u n c t i o n a l  meaning  membrane a r o u n d  maturachanges  pattern of these  unknown.  cell  r a t and  discovery  region  the other  either  is still  The  dog,  to the development  t h e neck  between  might  However,  Bedford  ram,  the base of the  or a consequence  shape which  Fulka  through the  o f s p e r m a t o z o a was  differences  The C e l l  left  1965b) i m p l i e s  physiological the s t a t e  1965;  However, an u l t r a s t r u c t u r a l  (Bedford,  1969a),  microscope occurred  these changes  the presence of the d r o p l e t  spermatozoa  Glover,  stallion,  a marked  a light  droplet  related  Phillips,  N i c a n d e r , 1971),  1971),  as t h e y p a s s  by  and  F o o t e , 1968;  and  the  o b s e r v e d i n the acrosomal  Because  measured  (1963a)  and  (Bedford  i n t h e human  the c y t o p l a s m i c  Bedford  Paufler  species:  Fawcett  ( F a w c e t t and  r a t (Bedford,  modification  (Bedford,  acrosomal after  1965), boar  of spermatozoa  epididymal mouse  1966;  white-tailed  1963;  1965b; Gaddum and  and  bull  i n several  the  of  86 acrosome  o f caput spermatozoa  move down related during the  the epididymis  this  lying  structure,  Stefanini  i n membrane  structure cauda  electron  epididymis)"but  I n membrane  observed felt  be  Also  amount, o r an  However, Zamboni and  configuration Jones's  membrane  t h e changes  d i d n o t f i n d any of caput  (1971)  and c a u d a  f i n d i n g s on  of the boar i n t h e membrane  due t o t h e f i x a t i v e s  of the s t a i n i n g  properties  properties  the c e l l  membrane may  spermatozoa,  (ii)  ( i i i ) fusion  spermatozoon  properties  (Bedford,  (caput  used f o r  (Bedford,  1965a; M a r t a n  as s p e r m a t o z o a  i s followed penetration  of the c e l l  1962a),  move  and H r u b a n ,  1970)  through the  change  i n t h e membrane.  by ( i ) c a p a c i t a t i o n o f  o f t h e e g g membrane, membrane  (Saunders, 1970),  property  (Glover,  1963b) and s u r f a c e -  account f o r the s u r f a c e  fertilization  membrane  head  microscopy.  epididymis  and  (1965b)  They  were n o t due t o t h e s o u r c e s o f s p e r m a t o z o a  electrophoretic  Since  also  o f t h e membrane m i g h t  o f t h e head  suggested that  Modification  adhesive  (1969a)  and g u i n e a p i g .  permeability.  of the r a b b i t .  spermatozoa  Bedford  d e c r e a s e i n t h e volume o f t h e u n d e r -  membrane  ultrastructure  as s p e r m a t o z o a  o f t h e sperm  and Zamboni e_t aJL. (1968)  in cell  spermatozoa  of  separation  o r an i n c r e a s e  (1968)  difference  and P h i l l i p s  i n the c h i n c h i l l a  by a p r o g r e s s i v e  alternation  or  Fawcett  the i n c r e a s i n g  caused  and a r e e j a c u l a t e d .  to the osmotic status  maturation.  same c h a n g e  that  the  change  becomes s w o l l e n  o f t h e e g g and  i t i s possible  of spermatozoa  i s normal  that  changes i n  f o r their maturation.  87 d. as  The the  Middle  cytoplasmic Anberg  1948). ment and  observed  the  reported  tails  the  epididymal  of  Specific  was  increased  1966). the  Foote  The  during  1952;  Lindahl  passed  ( 1 9 6 8 ) and  low and  and  the  l o s s of  the  droplet.  also  reported  An  that  the  (Lindahl  and  increase Lavon e t  not  caput  and  original  source  testis.  (Lindahl Lavon  the  per  (1970).  and  loss  of  latter  has  1952).  reported  head was  cells  e_t a l . ,  the  Kihlstrom,  i n the al.  the  Branton  (1947)  that  (1965) l a t e r the  decreased  Salisbury  c h a n g e was  provided  (1975)  spermatogenic  1965;  of  the  (1934),  the  ripening  for this  increase  (1968) d i d  and  g r a v i t y of  g r a v i t y of  by  during  ElWishy  Foote  Thunqvist,  Thunqvist  specific  arrange-  through  spermatozoa  Branton  droplet,  density  the  Hart,  (1962b)  spermatozoa from  process of  reason  of  m a t t e r was  heads but  s p e r m a t o z o a was  Specific the  suggested  However, L i n d a h l  Almquist  I g b o e l i and  abnormal  Gravity.  a comparatively  the  abnormal  addition,  r e s i d u a l cytoplasmic  increase  Amann and  e_t a l . (1973) c o n c l u d e d  f.  mitochondria  However, L a g e r l o f  morphologically  Kihlstrom,  of  transit.  In  and  spermatozoa.  a d i f f e r e n c e between  Bedford  (Rao  abnormal  (1947) and  straighter  some c h a n g e s i n t h e  spermatozoa  and  and  number o f  cauda e p i d i d y m i s . and  of as  Paufler  that  such  i n human  decrease  Salisbury  find  moves d i s t a l l y  Spermatozoa.  epididymis.  and  becomes t h i c k e r  (1957) o b s e r v e d  or broken  during  droplet  transit  Abnormal  bent  This  structural organization  epididymal  e.  Piece.  not cent  an caused of  dry  by  g.  Reflecting  the  s p e r m a t o z o a n head d e c r e a s e s  capacity  to  Capacity  light  to L i g h t .  As  with  ripening,  increases (Lindahl  Chemical  Changes  a.  Chemical  Composition.  Although  appearance of  the normal  spermatozoan  from  of  changes  time i n the  spermiation u n t i l  spermatid protein  condenses,  positive  m a t e r i a l (DNA)  Setchell  there  i s no  et  (DNA)  alteration  has  chromatin  ejt a l . , 1973;  the  (Fleistrich  and  and  of  the b a s i c  take the.  nuclear  e t a l . , 1966).  1966,  the  and  1971;  Feulgen Gledhill  light  et a l . ,  Because  maturation  Hulin,  I t has  a  DNP  and  i n the  the  been  the  and  nuclear 1971;  1975 ) s u g g e s t  nuclear  through  for  1971)  Bedford,  Marushige,  complex  1966).  Gledhill,  bonds  (Lavon  qualitative  of b i n d i n g s i t e s  1969;  M a r u s h i g e and  spermatozoa  as  (Gledhill,  (Calvin  of  absorption  b i n d i n g of the  of d i s u l f i d e  et, a l . , 1 9 7 6 ) .  nucleus  interpreted  stabilization  passage of  of  significant  complex  reduction in  a p r o g r e s s i v e decrease  increment  the  constant  in total deoxyribonucleic  by  o f e u t h e r i a n mammals  condensation during  been  during epididymal  actinomycinD(Brachet  Calvin  change  determined  this  remains  O r g e b i n - C r i s t , 1969).  i n the c o m p o s i t i o n  considerable  nucleus  (Gledhill  (Gledhill,  e t a l . , 1969;  content  Furthermore,  As  composition  quantitative  a l . , 1971),  t o DNA  the  reflecting  structural  fertilization,  there i s a continuous  1966;  acid  passage.  changes q u a l i t a t i v e l y  Subsequently  the  d e o x y r i b o n u c l e o p r o t e i n (DNP)  place during epididymal  the  in  e t a l . , 1952 ) .  3.  the  the water c o n t e n t  chromatin epididymis  observed  that  a  disulfide and  The  during  content of  ram  ( Q u i n n and  e t a_l. , 1 9 7 0 ) ,  and  during  rabbit  spermatozoa  o f sodium  passage  reported  and  during  and mouse reports  by  on  changes  1907).  ejaculated  were d e t e c t e d  ejaculated  ejt a_l. ,  1964).  1967; by  A  spermatozoa  tract  has  also  been  investigators.  However,  of r a b b i t  Glover,  The  transit  there  are  phosphatase spermatozoa  1972).  phosphatase  from  could  1975)  (Bavdek  i n the  and  Glover,  Indophenol oxidase a c t i v i t y  hand,  phosphatases  n o t be  spermatozoa  epididymis activities  e l i c i t e d , w h i l e they  (Gordon,  1973).  m e t a b o l i c aspects of epididyrnal  spermatozoa  the  conflicting  activity  pass through the  the other  i n cauda  Acid  tract  i n t h e r a t ( T e r n e r e_t a_l. ,  in alkaline  On  i n enzyme a c t i v i t y -in  decreases during  spermatozoa  Metabolism.  in  accumulated  Scott,  contents i n  and  transit  Scott  p l a s m a l o g e n was  several  decreases w h i l e spermatozoa (Ostwald,  epididyrnal  through the r e p r o d u c t i v e  1975).  droplet  Moniem and  spermatozoa  phospholipid  1967a;  Variations  epididymis  (Poirier,  cytoplasmic 1970;  While  (Dawson and  passage  of spermatozoa  to cauda  of  (Bedford,  1967).  h a v e been r e p o r t e d activity  transit  protein  the r e p r o d u c t i v e  Enzyme D i s t r i b u t i o n .  caput  and  structure  m a t u r a t i o n (Lavon e t a l . ,  White,  chloride  through  (Quinn,  spermatozoa  c.  lipid  choline  i n the t a i l  epididyrnal  plasmalogen decreased during  decrease  in  formed  T e r n e r e t a l . , 1975).  b o a r and  Lavon  b.  material  total  1971;  choline  rat  are also  estimated to decrease during  1970,  the  links  perinuclear  1975). was  cross  have been m e n t i o n e d  already  and  i n Chapter II  90 (pp. 53-55).  In t h i s  the metabolism Wu  section,  of t e s t i c u l a r  might  t h e Embden-Meyerhof  Ram  appreciable  (Murdoch  and  Moreover,  The  White,  inositol  testicular  by  hand,  utilized  Nevertheless,  or e j a c u l a t e d  but  Voglmayr  synthesis  was  and  acids  1966,  White,  spermatozoa,  1967).  only i n  1971).  of exogenous carbon i n t o  spermatozoa  spermatozoa  testicular  et a l . ,  the  spermatozoa.  not e j a c u l a t e d  demonstrated  (Voglmayr  testicular  shunt  glucose v i a  amounts o f g l u c o s e t o amino  of i n c o r p o r a t i o n  than e j a c u l a t e d other  testicular  1968;  i s on  testicular  denied the o c c u r r e n c e of  spermatozoa,  spermatozoa  rate  pholipids  1970)  in either  testicular  spermatozoa  scheme o f g l y c o l y s i s .  e_t a _ l . (1967,  pentosoe c y c l e  bovine  m e t a b o l i z e g l u c o s e v i a the pentose  pathway, w h e r e a s e p i d i d y r n a l  Voglmayr  the emphasis  spermatozoa.  e_t a _ l . (1959) s u g g e s t e d t h a t  spermatozoa  convert  therefore,  (Scott  spermatozoa  was  3 to 4 times  e_t a l . , 1 9 6 7 ) . incorporated  phos-  greater  On  much  the less  14 C  from g l u c o s e i n t o  volatile  ejaculated  spermatozoa  largely  formate r a t h e r  as  ejaculated spermatozoa  spermatozoa failed  fatty  i n that  and  differed  the i n c o r p o r a t e d  t h a n as  (Scott  acids  the a c e t a t e  et a l . ,  to hydrolyze  1967).  choline  Ram  spermatozoa  and  4.  Physiological  a.  Fertility.  spermatozoa  that  by  testicular  or ethanolamine  b u t b o t h were m e t a b o l i z e d by Dawson,  appeared  formed  phosphoglycerides, (Scott  *C  ejaculated  1968).  Changes  The  most  important f u n c t i o n a l  takes place  from  i n the e p i d i d y m i s  change of i s the  91 acquisition It  was  rat  of  shown  in several  (Blandau  (Nishikawa  fertilizing  and  and  and  Koefoed-Johnsen,  and  Foote,  1968;  ( H o r a n and boar  incidence 1967a), Crist,  of  the  Bedford there  1966;  with  epididymal  (1975) r e p o r t e d  was  no  1976)  statistical  embryos c o n c e i v e d  with  1966a,b,  1967b;  Fulka  1967a,b;  Griel,  1966a;  1974) The  rate  (Orgebin-  1 9 7 1 ) , and  On  embryonic  after  the  implantation  contrary,  had  difference in mortality with  and  Orgebin-Crist,  were o b s e r v e d  e j a c u l a t e d or  hamster  a l . , 1976).  cleavage  once  Paufler  1971),  and  spermatozoa.  that,  1931),  rabbit  Orgebin-Crist,  Smidt,  (Young,  1975),  Hunter et  i n the  pig  transit.  (Hoppe,  (Amann  (Bedford,  delay  ( H o l t z and  guinea  Orgebin-Crist,  1976;  Cummins and  epididymal  Orgebin-Crist,  1972 )', b u l l  striking  insemination  the  Bedford,  p o l y p l o i d ova  1968;  mortality  1952;  Smidt,  during  1 9 6 1 ) , mouse  Cummins and  Bedford,  ( H o l t z and  species:  Rumery,  Waida,  ability  occurred,  between  epididymal  spermatozoa. The, c o n t r o v e r s y , brought alone  about  (van  by  der  the  Glover,  (Tournade,  1913;  Koef o e d - J o h n s e n ,  on  the  1897;  has  Nicander,  Jolly,  or  Redenz, 1966;  aspects  Young,  quality Young,  by  1924;  Bedford,  of  the  1931;  epididymal  von  Lanz,  1975;  support  of  spermatozoon 1962b;  influence  1924;  Fulka  Orgebin-Crist However,  Ramos, 1973;  the  epithelium  1973;  and e_t a l . ,  observations  epithelium  and  1929a; Dyson,  are  Glover,  the  epididymal  Basrur  changes i n the  f o r many y e a r s .  1957a,b;  androgenic  1905;  1893;  1965)  been debated  dynamic  1 9 7 5 ) , on  intrinsic  Stricht,  Gaddum and  1975)  whether maturation  (Hammar,  Hamilton, (Marshall  Orgebin-Crist  and  et a l . ,  92 1975),  on  obtained the  different from  the t e s t i s  formation and  changes  during  indicate  B e d f o r d , 1971) the passage  (Bedford,  that  spermatozoan of  Paufler  and  of d i s u l f i d e  (Calvin  epididymis  c h e m i c a l c o m p o s i t i o n s of  on  of spermatozoa  Foote  for rabbit  fertile  i n the e p i d i d y m i s ,  species  of animals.  parts  of the e p i d i d y m i s  different  environmental conditions  corpus epididymis Fulka  and  of the r a b b i t  Orgebin-Crist,  the  mouse  and  B e d f o r d , 1972),  (Hoppe,  a l . , 1976).  physiology might b.  and  Species  have  cent  or i n the cauda  sites  Among s e v e r a l  (Mann, 1 9 6 4 ) . of p r o g r e s s i v e l y  1971).  the  1967a; epididymis  1976;  i n t h e anatomy Nicander,  of  (Horan Hunter and 1971)  of m a t u r a t i o n changes.  m a t u r a t i o n changes,  functional  connection  Although the c o r r e l a t i o n motile  quite  1967b;  hamster  Smidt,  and  of  1966a,b,  1971)  and  fully  Nicander,  Orgebin-Crist,  (Glover  extrinsic  takes place i n  and  1973),  remark  f o r several  lower h a l f  (Bedford,  differences  and  probably require  i n the  p i g (Holtz  t h e most d i r e c t  good  (Glover  r a t (Dyson,  account f o r diverse  fertility per  1975),  of  t o become  1966;  of the e p i d i d y m i s  Motility.  might  appear  Koefoed-Johnsen,  Cummins and  et  first  intrinsic  to hold  and  1971),  the c o n c l u d i n g  spermatozoa  different  the  site  spermatozoa  m a t u r a t i o n o f mammalian  spermatozoa  Bedford,  both  appears  membrane  through  i s an e s s e n t i a l  (1968), that  were r e q u i r e d  Fertile  and  on  i n the n u c l e u s  spermatozoan  Nevertheless,  factors  The  and  1967b; C o o p e r  maturation.  (White, 1973b),  cross-linking  the e p i d i d y m i s  and  epididymis  fluids  spermatozoa  and  motility with between fertility  the  93 was  estimated  Linford  et  t o be v e r y  fertilizing  (Young,  1929a;  In  t h e dog  1913;  species,  reported  motility. prior  that  Spermatozoa  1929a;  rabbit  Orgebin-Crist, Bhattacharya,  Yochem,  1930),  motility  1949;  Mukherjee  (Young,  This  1913),  lose  motility  increases has  been  as found  r a t (Tournade,  guinea p i g (Tournade,  ( T o u r n a d e , 1913;  1967a), b u l l  of  may  1941).  spermatozoan  Tournade,  1963),  the n o n r e t u r n  to c e s s a t i o n  Salisbury,  (Hammar, 1897;  1929a),  1929a;  and  and H u r s t ,  pass through the e p i d i d y m i s .  Young,  Young,  with  capacity  Asdell  several  spermatozoa in  (Kelly  a l . (1976) r e c e n t l y  v a l u e s were c o r r e l a t e d their  low  B e d f o r d , 1966a,  1929a,  Amann and A l m q u i s t ,  Mukherjee  1 9 6 2 a ) , ram  and B h a t t a c h a r y a , 1 9 4 9 ) ,  and human  1913;  1967b;  and (Young, (Bedford,  1973). Recently, in  the changing p a t t e r n  the e p i d i d y m i s  released  from  vibratory Those  the t e s t i s  or c i r c u l a r  released  progression, epididymis rotation  has b e e n  and most  Gaddum, 1968;  neck  region  and  1961;  1929a; Fulka  mink  organelles  cauda  longitudinal Waida,  Koefoed-Johnsen,  1974).  In a c c o r d a n c e w i t h tail  from the  N i s h i k a w a and  and  Griel,  movement o f e j a c u l a t e d  various  develop forward  and F o o t e , 1968;  Amann and  (Onstad, 1967).  progress.  f o r w a r d movement w i t h  Young,  Igboeli  show weak  forward  spermatozoa r e l e a s e d  Rumery,  et. a l . , 1973;  progressive  no  motility  Spermatozoa  caput epididymis  movement w i t h  ( T o u r n a d e , 1913;  1966;  40-80%  and  show p r o g r e s s i v e  B l a n d a u and  of  much e m p h a s i z e d .  from the c o r p u s e p i d i d y m i s  1952;  Bedford  of spermatozoan  Bedford, The  per  1973; cent  spermatozoa i s m a t u r a t i o n , the  seem t o become  less  94 flexible  and  disulphide rigid  c.  are s t a b i l i z e d  bonds,  vital  stain  spermatozoa.  stain  Brochart unstained  ruminants. between marked  (1942)  test  introduced  a  for ejaculated  membrane  permeability,  whereas  active  not s t a i n  (Lasley  et. a_l. , 1942;  or  potentially Amann  1962b). and D e b a t e n e  (1953) r e p o r t e d  from  However, o t h e r  Salisbury,  1947;  and F o o t e , 1 9 6 8 ) .  (1962b)  suspected  that  to d i f f e r e n t  from  f o u n d no  increase  epididymis  such  of  difference  t h e c a p u t and c a u d a  and  spermatozoa from the corpus Amann and A l m q u i s t ,  Therefore,  1962b;  Amann and A l m q u i s t  these c o n f l i c t i n g staining  a fourfold  the c a p u t t o cauda  workers  a decrease i n unstained  due  B e d f o r d , 1975).  readily,  Igboeli  been  e t a_l.  as a q u a l i t y  spermatozoa  and  ejt _al_. , 1973;  Lasley  spermatozoa c o l l e c t e d  (Branton  of  s p e r m a t o z o a d e v e l o p a more  of increased  s p e r m a t o z o a do  and A l m q u i s t ,  In  eosin  Because  spermatozoa  active  (Bedford  Property.  using  the e s t a b l i s h m e n t  t h u s mature  t y p e o f movement  Eosinophilic  dead  and  by  results  techniques  might  have  or sampling  differences.  d.  Temperature  are  more r e s i s t a n t  testis  contrary,  the  testis  Wales,  Spermatozoa  to high  or ductus deferens  the  1930;  Shock.  resistance  1961;  et al.,  (Yochem,  Voglmayer  1930;  of spermatozoa  1942;  the cauda  epididymis  temperature than those from  t o cauda e p i d i d y m i s  Lasley  from  and  Bialy  Young, to cold  1931).  e t a_l. , 1967;  Smith,  1959;  Setchell  On  d e c r e a s e s from  ductus deferens  and  the  (Yochem, White  et a l . ,  and  1969).  95 e.  R e s i s t a n c e . bo  of  spermatozoa  harmful to  Alkalinity  to a l k a l i n i t y ,  alkaloids  Net  charge  spermatozoa  epididymis 1971), and This  of  decreases  Bedford, appears or  (Cooper 1971;  Bedford,  1973;  net  1971),  a modification  nature of  ionized  Property.  spermatozoa  head-to-head  bundles  pig  and  1957a).  and  This  mink  1975).  large  Bedford, (Cooper  of  cell  lead  of the  to  head-to-  negative  and  (Onstat,  Lindahl  head-to-head  i n the  guinea  (Nicander,  v/as a l s o  reported in  1967). changes  in specific  i n net  r e g i o n s of  normal  serum  w h i l e ADP  charges  the  i n the p r e s e n c e  (1.968) r e p o r t e d t h a t  agglutination,  moving  rouleaux-like  rabbit  i s i n c r e a s e d as  I t i s enhanced  randomly  transit  and  o r s m a l l amounts o f  Furthermore,  and  agglutination  spermatozoa  surface.  cations  increased  and  the  capacitation  into  H o l l e n b e r g , 1963)  in antigen distribution  divalent  i n the  human  T h i s may  during epididymal  adhesiveness  spermatozoan  deferens.  et a l . , 1973).  over  Individual  aggregate  T h i s head-to-head  ejaculated  testis  i s obscure.  Agglutinating  (Fawcett  the  i n the d i s t r i b u t i o n  groups  passage.  and  negative surface  Bedford  i n spermatozoan m a t u r a t i o n ,  testicular  and  i n the ductus  a u t d a g g l u t i n a t i o n . However, t h e r o l e  fertilization  g.  (Yochem, 1930)  i n c r e a s e s from  The  Bedford,  membrane d u r i n g e p i d i d y m a l  charge  resistance  ( B e d f o r d , 1963b; C o o p e r  and  to r e f l e c t  the  The  increases during maturation  the r a b b i t  monkey  charges  head  acidity  Negative Surface Charge. of  Acidity.  ( M e t a l n i k o v , 1911)  cauda e p i d i d y m i s , then  f.  and  of  (Bedford, ATP  lowered  the  96 frequency. failed serum of  However, a n t h r o p o i d  t o show h e a d - t o - h e a d (Bedford  human  autoagglutination  e t a_l. , 1973 ).  spermatozoa  monkey and human  The  spermatozoa  i n normal  autoagglutination  seems t o be r e l a t e d  to s t e r i l i t y  (Wilson,  1956).  h.  Contact A b i l i t y .  region  of the r a b b i t  because  of lack  to e s t a b l i s h Perhaps  epididymis  of m o t i l i t y ,  contact  during  Spermatozoa  passage  o f spermatozoa  maintain  contact  (Horan  remains  unknown.  from  to f e r t i l i z e  but a l s o  because  surface  develops t h e i r  w i t h t h e ovum..  not only  (Bedford,  ability  Although this  (Amann and G r i e l ,  and B e d f o r d , 1 9 7 2 ) ,  the proximal  they are unable  through the epididymis,  confirmed i n the b u l l  hamster  fail  w i t h t h e ovum  membrane  also  collected  1966a).  the c e l l t o i n i t i a t e and phenomenon was  1974) and S y r i a n  the nature of t h i s  contact  97 C.  MATERIALS  1.  The P r e p a r a t i o n o f M a t e r i a l  The  AND  METHODS  reproductive tracts  investigation  were t h e same  Small  pieces of tissue  parts  of the epididymis  minks  ( F i g . 38).  these  tissues  solution  2.  from  from  (caput, corpus  the t e s t i s  such  f o r enzyme  sample. The light drop Eosin  Five  spermatozoan  mating  8-12  test  and V i a b i l i t y  study,  semen was  by a 1 ml s y r i n g e f i t t e d Since  microscope  different  stained  and d r i e d  count  with  t h e same  citrate  with a  collected as a  test  study.  o f 300 c e l l s  test,  amount o f s t a i n  (3%  Using  f o r each  a  Ahmad - u n p u b l i s h e d ) a light  microscope,  s a m p l e was c l a s s i f i e d  as  spermatozoa.  o f the. e j a c u l a t e d  Krebs-Henseleit-Ringer  For v i a b i l i t y  solution,  immediately.  and u n s t a i n e d  A portion  i n this  a t room t e m p e r a t u r e .  B i n 2.9% s o d i u m  total  collected  o f m o t i l e s p e r m a t o z o a were e s t i m a t e d by a  o f semen v/as m i x e d  a slide  Test  t h e mink e j a c u l a t e s  minks and p o o l e d  s a m p l e s were u s e d  percentage  immersing  localization.  (Ahmad e_t a l _ . , 1 9 7 5 a ) .  from  five  o r i n 1 ml o f 0.9%  a s m a l l amount o f semen, t h e semen was  individually  study.  and t h r e e  and c a u d a ) o f  m i c r o s c o p i c study  the ejaculated  tube  part of the  as f o r t h e u l t r a s t r u c t u r e  were s l i c e d  solution  after  Tract  i n 1 ml o f K r e b s - H e n s e l e i t - R i n g e r  f o r electron  vaginae  plastic  a  for this  The P r e p a r a t i o n o f E j a c u l a t e d Semen  For  on  used  the Reproductive  The s p e r m a t o z o a were o b t a i n e d by  either  sodium c h l o r i d e  from  solution  semen was washed t w i c e for electron  microscopic  with study  F i g . 3 8 . Schematic drawing o f a mink r e p r o d u c t i v e t r a c t to show the p o s i t i o n o f the v a r i o u s p a r t s (shaded r e g i o n s )  99 or  with  sperm-Ringer  solution  (Mann, 1946)  f o r enzyme  localization. 3.  The  Procedure f o r E l e c t r o n Microscopy  The  s u s p e n s i o n s of spermatozoa o b t a i n e d  locations  of the r e p r o d u c t i v e  tract  and  s p e r m a t o z o a were p r e p a r e d f o r e l e c t r o n cytochemical  s t u d y (pp.  Approximately tions i.e.  testis,  fine  heads  parts  f o r each  o f t h e head  morphological  length  of t o t a l  head,  apical  and m a i n  segments  postacrosomal thickness of and  The  statistically  by  on  ejaculated  f o r the  For each  section,  ( t h e sum  nucleus,  i n F i g . 39.  of the d i f f e r e n c e  changes  of v a r i a n c e  and  of  the of the segment,  acrosomal  The on  t h e m i d d l e p i e c e were  morphological  the a n a l y s i s  measure-  total  and  t h e means o f t h e s p e r m a t o z o a n  and o t h e r  sec-  f o r the e s t i m a t i o n  acrosome  as o u t l i n e d  droplet  (Armitage, 1971).  and  locations,  and  selected  segment,  significance  property,  meters measured  test  (5  membrane, t h e o c c u r r e n c e o f s w e l l i n g s  calculated.  ( p . 20)  of the acrosome), e q u a t o r i a l  sheath, a p i c a l  the c y t o p l a s m i c  staining  sample  and  changes.  anterior  were measured  the c e l l  microscopy  c a p u t , c o r p u s , cauda e p i d i d y m i s  ment o f s e v e r a l  ejaculated  micrographs of s a g i t t a l  semen X 5 r e p l i c a t e s ) were r a n d o m l y  other  washed  described  56-57).  50 e l e c t r o n  of spermatozoan  from  coherence t h e head also  in motility, head  para-  were d e t e r m i n e d  Newman-Keul's  100  acrosomal thickness  Fig.  39.  The p o s i t i o n s o f t h e head  measured  101 D.  RESULTS  1.  The V i a b i l i t y o f The  per cent  spermatozoa and  from  values  from  tract  tract.  spermatozoa  were c o l l e c t e d  as s p e r m a t o z o a  obtained  Testicular  from  from  and c a u d a  in  the t e s t i s passed  ejaculated  spermatozoa  t o each p a r t  i n spermatozoa  hand,  the cauda  was s i g n i f i c a n t l y of the  collected  the m o t i l i t y epididymis  spermatozoa  showed a  of  was h i g h e r  than  difference  increased  during  to the m o t i l i t y , sample was  significantly  ( v i a b l e ) spermatozoa  the caput e p i d i d y m i s .  c a p u t and c o r p u s e p i d i d y m i s ,  ejaculated  because  through the r e p r o d u c t i v e  of unstained  t h e r e was no s i g n i f i c a n t  Similar  s t u d y were  spermatozoa.  (p < 0.05) r a t e  spermatozoa  in this  T h e mean  from a n i m a l s not used f o r t h e r e p r o d u c t -  On t h e o t h e r  of e j a c u l a t e d  spermatozoa  and e p i d i d y m i s  The number o f m o t i l e  the t e s t i s .  and u n s t a i n e d  semen a r e shown i n T a b l e 2.  No a c t i v i t y was d e t e c t e d  spermatozoa  the  the t e s t i s  (p <C 0.05) f r o m  epididymis  higher  from  spermatozoa  s t a t i s t i c a l comparisons  study.  increased  that  removed  of e j a c u l a t e d  spermatozoa  from  of motile  the ejaculated  excluded  ive  Spermatozoa  Although  between  spermatozoa  t h e mean v a l u e s  passage  the per cent  than  from  of unstained  through the epididymis. of unstained  low as c o m p a r e d  to that  spermatozoa  o f t h e cauda  epididymis. 2.  Changes The  i n the Spermatozoan  mean v a l u e s  of several  Head  Length  portions  of spermatozoan  head  T a b l e 2. The p e r c e n t o f m o t i l e s p e r m a t o z o a and u n s t a i n e d s p e r m a t o z o a c o l l e c t e d f r o m s u c c e s s i v e p a r t s o f t h e r e p r o d u c t i v e t r a c t and e j a c u l a t e d semen  Epididymis Corpus  Testis Caput Motile /o  spermatozoa  (Mean ± SE)  Unstained spermatozoa % (Mean ± SE)  a* 0 a,b 52.0 ± 3.0  _. , , , Semen  E j  Cauda  b  c  d  1.0  20.0 + 2.4  84.0 ± 3.6  65.0 ± 2.0  c 31.2 + 2.7  b,c 39.8 ± 2.9  a 56.8 + 5.5  35.6 ± 3.9  7.2 t  •Means on t h e same l i n e w i t h t h e same s u p e r s c r i p t l e t t e r a r e n o t d i f f e r e n t d i f f e r e n t l e t t e r s a r e s i g n i f i c a n t l y • d i f f e r e n t , p < 0.05.  but with  lengths  from  ejaculated  spermatozoa  ejaculated collected The  collected  tract.  from  was  was  spermatozoa  smaller  significantly from  the  b e t w e e n a n i m a l s , and  animals  different  tract  were g r e a t e r  Although equatorial the  length  mean v a l u e s  each in  part  spermatozoa  from  was  spermatozoa  length  spermatozoa  length through  testicular  0.001) t h a n t h o s e  were  The  smaller  In  significant  from  of  tract.  The  the reproducspermatozoa.  and  corpus  spermatozoa,  i n spermatozoa t o be  greater  from than  equatorial length  than those of  that of  spermatozoa  tract.  spermatozoa.  greater  Even  appeared  of epididyrnal (p < 0.05)  though  d i f f e r e n c e , t h e mean v a l u e s  part  testicular  difference in  of postacrosomal r e g i o n  of spermatozoa each  appeared  were s i g n i f i c a n t l y  testicular  statistical  was  spermatozoa  of e j a c u l a t e d  significant  the t e s t i s .  the r e p r o d u c t i v e The  there  of e q u a t o r i a l length  from  of  i n the i n t e r a c t i o n  than that  between  the  of the e p i d i d y m i s .  of spermatozoa  no  of  length  of the r e p r o d u c t i v e  of the e p i d i d y m i s  ejaculated  of  there  length  parts  head  (p <  part  however,  acrosomal lengths  head  than that of  greater  differences  tive  total  no  tract.  various  parts  and  T h e r e was  of the a n t e r i o r acrosome o f  a n a l y s i s of variance,  and  tract  from d i f f e r e n t  the r e p r o d u c t i v e  length  anterior  3.  However, t h e t o t a l  spermatozoa  spermatozoa  the  of the r e p r o d u c t i v e  d i f f e r e n c e i n t h e means o f  reproductive  of  parts  semen a r e shown' i n T a b l e  significant between  four  there  that  no  of postacrosomal  to increase  of the e p i d i d y m i s .  was  than  The  during value  passage  of e j a c u l a t e d  Table  Part Total  3.  D i m e n s i o n a l changes at d i f f e r e n t o f t h e s p e r m a t o z o a n head  Testis  portions  Caput  Epididymis Corpus  Cauda  b t  „ . , 3 cu±a-  Signif icance Levels  a  e  a  b  e  m  e  n  head,  l e n g t h , um (Mean + SE )  5.86 + 0.05  5.80 ± 0.06  5.71 ± 0.05  5.83 + 0.05  Anterior acrosomail l e n g t h , um (Mean ,£ SE )  a . 6 1 ± 0.05  b 2.34 ± 0.05  b 2.25 + 0.04  b 2.29 + 0.04  1 . 4 7 ± 0.02  c 1.56 + 0.03  a,b 1.53 ± 0.02  b 1.55 1 0.02  1.46*0.02  p<0.05  a 1 . 7 7 ± 0.02  b 1.89 ± 0.03  b 1.93±0.02  b 1.99 ± 0.02  1.77 ± 0 . 0 2  p<0.05  Apical l e n g t h , ym (Mean SE)  a 0 . 9 0 ± 0.02  a 0.88±0.03  b 0.67 ^ 0 . 0 1  b 0.68±0.01  0.65+-0.01  p<0.00l  Nuclear l e n g t h , ym (Mean i SE) Acrosomal t h i c k -  a 4 . 9 5 i 0.04  a 4.89 ± 0.06  a 5.04 ± 0.04  a 5.15 ± 0.04  4.61 ± 0 . 0 4  p<0.05  a  b  b  b  ? f , ' +™ ^ (Mean I SE)  0 . 6 8 i 0.02  0.52*0.01  0.50 + 0.01  0.52 ± 0 . 0 1  0.55±0.01  p<0.001  Equatorial l e n g t h , jam (Mean ± SE) Postacrosomal l ym le en ng gt th h, , ym (Mean ± SE)  1  S S  2  a  5.25 ± 0 . 0 4  2.02 + 0.04  "Means on t h e same l i n e w i t h t h e same s u p e r s c r i p t l e t t e r a r e n o t d i f f e r e n t d i f f e r e n t l e t t e r s a r e s i g n i f i c a n t l y d i f f e r e n t as i n d i c a t e d .  p<0.05  p < 0.001  but with  105 spermatozoa  was s m a l l e r  Because in  this  the t o t a l  study into  postacrosomal acrosomal the  the t e s t i s  spermatozoa The  the  mean  testis  length  o f spermatozoa  a significant  compensated  postacrosomal length  from of  head  for a significant  during  the t r a n s i t  to the e p i d i d y m i s . remained,  values  Thus,  statistically,  of the apical  and c a p u t e p i d i d y m i s  apical  that  of cauda  length  spermatozoa.  increase of  of spermatozoa  the t o t a l  of s i m i l a r  length  the r e p r o d u c t i v e The  spermatozoa  from  greater  hand,  to  the nuclear during  The n u c l e a r  length  than those o f spermatozoa  of spermatozoa  the e p i d i d y m i s .  the major  from  the t e s t i s  (p < 0.001) t h a n t h o s e o f  acrosomal thickness  In b r i e f ,  was s i m i l a r  significantly  tract.  less  epididymis.  tract.  acrosomal thickness  was s i g n i f i c a n t l y  greater  was  values.  of spermatozoa of  spermatozoa  d i d not change  spermatozoa  lengths  were.significantly larger  On t h e o t h e r  through the r e p r o d u c t i v e  ejaculated  from  of ejaculated  of spermatozoa  passage of  length  was d i v i d e d  decrease of the a n t e r i o r  (p < 0.001) t h a n t h o s e o f t h e c o r p u s and c a u d a The  spermatozoa.  t h e a n t e r i o r a c r o s o m a l , e q u a t o r i a l , and  length,  length  than those of epididyrnal  Ejaculated  than e p i d i d y m i d a l  s p e r m a t o z o a had spermatozoa.  changes i n the dimensions o f spermatozoan  head d u r i n g t h e i r t r a n s i t t h r o u g h t h e r e p r o d u c t i v e t r a c t were t h e decrease  of the a n t e r i o r acrosomal  thickness without  and t h e i n c r e a s e  alteration  length  and a c r o s o m a l  of the postacrosomal  of the t o t a l  head  length.  length  10G 3.  Changes  i n the Morphology  Because in  only  spermatozoa  tract,  very  during  spermatozoa  fine their  of the Spermatozoan  Head  m o r p h o l o g i c a l changes  take  passage  collected  The  acrosome.  successively the  was  of q u a l i t a t i v e  noted  (Figs.  size  of n u c l e a r  from  the t e s t i s  ejaculated In  semen  changed  (Figs.  (Figs.  (Figs.  as a p y r i f o r m  (Figs.  the o v a l  form  shape  Several  ( F i g . 50)  (Figs.  t h e n u c l e u s and  and  The  number  bizarre  and  an  and  and  in 40, 41)  ejaculated  forms  of  acrosomes  ( F i g . 52)  spermatozoa  T h e r e was  than i n corpus  spermatozoa  and  (Figs.  S-shape  the acrosomal r e g i o n  40, 49, 54)  ejaculated  transit,  epididymis  caput epididymis  51, 53).  43)  40-44).  i n corpus, cauda,  42-44).  caput epididymis  (Fig.  case the acrosomal  form o f the acrosome  the  (Figs.  A  43, 4 4 ) .  spermatozoa  spermatozoa  3).  of the spermatozoan  were o b s e r v e d i n t e s t i c u l a r  between  of  aspects of  49, 52) t o t h e c a u d a  t h e t e s t i s and  to a s l e n d e r  spermatozoa such  from  dense  (Table  i n other  In t h i s  sequence  sagittal sections,  spermatozoa  change  parts  vacuoles s i g n i f i c a n t l y decreased i n  a c c o r d a n c e w i t h the above i.e.  ejaculated  40-44).  m a t r i x became more e l e c t r o n and  appearance.  passed through v a r i o u s  and were e v e n t u a l l y  sequence  acrosome  microscopic  and  acrosomal dimension decreased  as s p e r m a t o z o a  epididymis  similar  The  reproductive  from t e s t i s , e p i d i d y m i s  semen, d i d n o t d i f f e r i n g e n e r a l  a.  through the  place  more  from  space  in testicular ( F i g . 42),  ( F i g . 44).  cauda  107 b.  The c e l l  membrane.  membrane was u s u a l l y (Fig.  44) t h a n  The e x t e n t  of separation  more p r o n o u n c e d  in testicular  of acrosomal  i n ejaculated  or caput  spermatozoa  spermatozoa  (Figs.  41,  49, 50, 54, and T a b l e 4 ) . The  ratio  spermatozoa  of the apposed  was s i g n i f i c a n t l y  cell  membrane o f  greater  (p < 0.01) t h a n t h o s e o f  epididyrnal  spermatozoa.  Although there  difference  between p a r t s  of the epididymis,  the  apposed  epididymis  membrane r a t i o  than cauda (p the  Ejaculated  of spermatozoa  spermatozoa  spermatozoa.  < 0.05) o f s e p a r a t e d  membrane  testis.  Ejaculated  spermatozoa  membrane  than cauda  spermatozoa.  T h e r e was no s i g n i f i c a n t damaged membrane d u r i n g reproductive  tract.  than  may r e m a i n  c.  though  was s e p a r a t e d  of the  membrane  increase collected  to that  had s t i l l  more  from  from the  separated  i n the i n c i d e n c e o f  o f spermatozoa  spermatozoa  through the  had more damaged  epididymis.  Cell  membrane  It i s well  t h e membrane a l o n g  o r damaged known t h a t  (Figs.  other  t h e p r o c e s s o f sperma-  micrographs, the m i c r o s t r u c t u r e  demonstrated  (Figs.  parts  41, 43, 44, 5 5 ) .  and s p e r m i o g e n e s i s o c c u r i n t h e t e s t i s .  the e l e c t r o n  immature  apposed  compared  t h e cauda  part  moved t h r o u g h t h e  had l e s s  difference  Ejaculated  sheath even  Spermatism.  was c l e a r l y  i n each  i n spermatozoa  the passage  those from  spermatozoa  tocytogenesis in  t h e mean v a l u e s o f  t o be c o n n e c t e d w i t h t h e e d g e o f t h e a c r o s o m e and  postacrosomal of  significant  T h e r e was a s i g n i f i c a n t  c o r p u s and cauda e p i d i d y m i s  membrane  was no  were d e c r e a s e d a s s p e r m a t o z o a  epididymis.  testicular  45-48).  As shown  of spermatids  Occasionally,  s p e r m c e l l s were o b s e r v e d i n t h e c a p u t  epididymis  T a b l e 4. C h a n g e s i n t h e c e l l membrane c o n f i g u r a t i o n and i n t h e o c c u r r e n c e o f s w e l l i n g s and t h e c y t o p l a s m i c droplet  P  a  r  Testis  t  C  a  p  u  Epididymis Corpus  t  Cauda  Eiaculated Semen  Significance Level  16.2±  p<0.01  C e l l membrane configuration: Apposed  acroso-  % (MeJn ± SE) a  b  a  a ! S  9  8.0i  1.3  Separated aero^(Mea^+^SE) Damaged  6  '  0  ±  Q  ±  °*  6  f ?i  Q  ^  6  4 4 . 7  + 2 1 . 2  % (Mean I SE) Postacrosomal ,,. a S ?M + ctr> 1 8 . 8 ± 15.9 % (Mean ± SE) Occurrence o f cyto^ o /  %  a  / M  9  1  C  -  6  ±  1  1  '  2  +°^t  t S  26.4+5.5  3.2  . 41.7 + 11.5  3  3 . 6 + 1 8 . 3  , a,b  +  W  P  9  9.1  ^  a 1  33.8+  2 4 . 5  +  7 . 2  4 8 . 8 ± 7.5 a  2 4 . 8  ±  54.0 ±  9 . 4  8.2  p<0.05  2 9 . 9 ± 1 0 . 1  -  Acrosomal  d  56.8±19.3  a 1  b  acroso-  mal membrane % ( M e a n + SE ) Occurrence of swellings:  W  b  6 2 . 2  +  5 . 8  , b  a 2 4 . 5 ±  1 3 . 3  8 6 . 1  +  5 . 5  3 0 . 9 ±  1 6 . 4  9 2 . 5  +  3 . 9  8 7 . 6  9 0 . 0  ±  1  4 . 6  ±10.3  0 . 8  ±  0 . 8  9 8 . 4  ±  5 . 0  4 . 7  p < 0 . 0 5  ^  , b 9 8 . 3 ±  5 . 2  p < 0 . 0 1  ^  b 2 1 . 6  N  • , b  , b  a  p < 0 . 0 5  ^  b 0 . 8  +  0 . 8  0 . 4  ±  0 . 5  (Mean I SE)  * M e a n s o n t h e same l i n e w i t h t h e same s u p e r s c r i p t l e t t e r a r e n o t d i f f e r e n t b u t d i f f e r e n t l e t t e r s a r e s i g n i f i c a n t l y d i f f e r e n t as i n d i c a t e d .  p < 0 . 0 0 1  ^ with  109 (Figs. in  d.  51, 5 3 ) .  spermatozoa  The  However, from  swelling.  acrosomal  spermiogenesis  regions posterior  Testicular  swellings  incidence (p  of  < 0.05)  tozoa.  On  swellings  was  found  and  spermatozoa  was  passed  through  had  no  trend  definite  perforatorium  remained  4.  c o n t e n t was  Changes Owing  of  the spermatozoan tail  the  movement o f  There  was  a l l parts intact  could  tail,  n o t be  spermatozoa  collected  The  cytoplasmic droplet  head  the  time of  ejaculation.  sperma-  postacrosomal (p <  as  and  0.01)  from Almost  swellings. length  spermatozoa  subsequent  i n c a s e s where  of g e t t i n g  (Figs.  piece  from  the  entire  14, 5 7 ) .  Tail  longitudinal  only obvious  sections  change  t h e neck  ( F i g . 23, 58 and  (p <  the t e s t i s found  the  m o r p h o l o g i c a l changes  The  difference  was  cauda  of the Spermatozoan  detailed  from  increased  of the r e p r o d u c t i v e t r a c t ,  even  of the middle  (Table 4).  i n t h e o c c u r r e n c e and  observed.  a significant  and  types of  the c y t o p l a s m i c d r o p l e t  end  more  spermatozoa.  s e p a r a t e d o r damaged  to the d i f f i c u l t y  the d i s t a l  both  spermatozoan  i n the Morphology  the  to  cauda  the r e p r o d u c t i v e t r a c t Through  acrosomal  significantly  increased  and  spermatozoa  ejaculation.  spermatozoa  the o c c u r r e n c e of  to corpus  p e r f o r a t o r i u m i n the  heads had  to corpus  significantly  caput  ejaculated There  of  caput  observed  the e p i d i d y m i s , the  s w e l l i n g s was  t h e o t h e r hand,  testicular all  acrosomal  from  spermatozoan  through  not been  to the caput e p i d i d y m i s .  than, those of caput  However, d u r i n g t h e t r a n s i t  has  of was  portion Table 4).  0.001) b e t w e e n  and  from  virtually  the e p i d i d y m i s .  to disappear  by  110 The  scroll  spermatozoa fibrils  (Figs.  7, 8 ) .  were a l m o s t  and  spermatozoa  5.  Changes The  from  was o b s e r v e d  to that  between e j a c u l a t e d tract  spermatozoa  (Figs.  14, 5 6 ) .  Activity  o f enzyme d i s t r i b u t i o n  the r e p r o d u c t i v e  similar  and e j a c u l a t e d  and number o f s a t e l l i t e  the reproductive  i n t h e Enzyme  pattern  The form  similar  from  i n cauda  tract  i n mink  was, a s d e s c r i b e d  i n spermatozoa  from  spermatozoa  i n Chapter I I ,  t h e cauda e p i d i d y m i s .  However, t h e i n t e n s i t y  o f t h e enzyme a c t i v i t y i n t e s t i c u l a r  spermatozoa  than  with  that  i n epididymal  t h e e x c e p t i o n o f DOPA o x i d a s e .  (acid LDH,  was weaker  and a l k a l i n e  phosphatases,  NADH d i a p h o r a s e ) a p p e a r e d  spermatozoan  passage  through  spermatozoa  Some enzyme a c t i v i t i e s  G - 6 - P a s e , A T P a s e , MDH,  t o be i n c r e a s e d  d u r i n g the  the e p i d i d y m i s , while the other  enzyme a c t i v i t i e s ( 5 - n u c l e o t i d a s e ,  ADPase, e s t e r a s e ,  oxidase,  Although  IDH) r e m a i n e d  (5-nucleotidase, showed tive  similar  tract  activities alkaline following  unchanged.  a c t i v i t y between  spermatozoa  and t h o s e i n e j a c u l a t e d  phosphatases, ejaculation.  esterase,  semen,  DOPA  some enzymes  DOPA o x i d a s e , LDH, IDH, NADH  (G-6-Pase,  SDH,  diaphorase)  from  the reproduc-  other'enzymes  SDH and p a r t i c u l a r l y  ADPase, A T P a s e , MDH) d e c r e a s e d  a c i d and  in  Table spermatozoa  5. ' C h a n g e s i n enzyme a c t i v i t i e s f r o m t e s t i s , e p i d i d y m i s and e j a c u l a t e d  Epididymis Enzyme  Testis Caput  Corpus  Cauda  E j aculated Semen  semen  Location  Phosphatases A c i d phosphatase A l k a l i n e phosphatase 5-nucleotidase G-6-Pase ADPase ATPase  ± ± + + +  ++ + + + ++ +  ++ + + + ++ ++ ++  ++ + ++ + ++ + ++ ++  Non-specific  ±  +  +  +  —  Head b a s e and m i d d l e piece  +  +  +  +  +  Head  Dehydrogenases Malate Succinate Lactate Isocitrate 6-phosphogluconic  + + + —  + + +, +  ++ ++ + +  -  -  ++ ++ + +  -  Head b a s e and Middle piece - ditto - ditto -  NADH  +  +  DOPA  Esterase  oxidase  diaphorase  *Key t o a b b r e v i a t i o n s :  - •  ++  ++  +  -+ ++ +  -  + + +  -  ++  Head  and t a i l - ditto Middle piece - ditto Head and t a i l - ditto -  and  Middle  +• weak r e a c t i o n ; + moderate r e a c t i o n ; ++ s t r o n g +++ v e r y s t r o n g r e a c t i o n ; - negative r e a c t i o n .  tail tail  piece  reaction;  112 Figures  40  t o 44.  during  of spermatozoan  passage through the r e p r o d u c t i v e  sequent Figure  Acrosomal changes  ejaculation 40.  The  large  oval  (sagittal  testicular  tract  and  sub-  sections).  s p e r m a t o z o a n head  showing  a c r o s o m e (AC) composed o f g r a n u l a r e l e c t r o n  l e s s dense m a t r i x . prominent.  The  perforatorium  There i s comparatively  between  the acrosome  several  vacuoles  and  heads  and  nucleus  (arrows).  The  postacrosomal swellings  (P) i s  wide  space  (M) w h i c h  has  acrosomal  (PS) a r e  (AS)  obvious.  30,000X. Figure  41.  The c a p u t s p e r m a t o z o a n head  oval-shaped contain cell Figure  acrosome.  electron  membrane  42.  The  less  The  (CM) . i s s e p a r a t e d .  corpus spermatozoan  the nucleus  swellings Figure  43.  and  are prominent.  The c a u d a  vacuoles have  The  disintegrating  The  the  27,000X. showing The  space  i s narrow  and  27,000X. heads  showing  acrosomes.  The  configurations  n u c l e u s becomes d e n s e  (arrows).  erected  acrosome  and  acrosome.  spermatozoan  membrane shows d i f f e r e n t cells.  and  head  acrosome  rounded cone-shaped dense  the  nucleus  dense m a t e r i a l  rounded cone-shaped dense between  showing  cell  between  and  has  few  postacrosomal swellings  membraneous m a t e r i a l  (MM).  27,000X. Figure  44.  long  The  ejaculated  s p e r m a t o z o a n head  rounded cone-shaped  acrosome.  showing  A l t h o u g h the  113 cell  membrane  acrosomal  (CM) i s s e p a r a t e d  surface,  the a p i c a l  a c r o s o m e and p o s t a c r o s o m a l t h e membrane. vacuoles.  The e l e c t r o n  25,000X.  from  the p e r i -  e n d (AE) o f t h e  sheath dense  (PS) a d h e r e nucleus  to  has s m a l l  114  115 Figures  45  to 49.  Sagittal  spermatozoa  i n the  Figure  The  45.  shaped  (GC).  The  showing evenly the Figure  Figure  48.  portions  distributed  The  inversed nuclear  and  fine  (MA).  (M)  granular condensed at 9,000X.  i n a c r o s o m a l phase  of the acrosomal  substances,  9,000X. of a spermatid  arranged  around  showing  the a x i a l  fiber  30,000X. i n maturation  phase  half-moon form acrosome.  (DG)  football-  c h r o m a t i n (GC.) and f o r m a t i o n o f  spermatid  ring  granules  showing  of the nucleus.  The m i d d l e p i e c e  (AF).  (AG)  spermatid  redistribution  mitochondria bundle  i n cap phase  The c h r o m a t i n has b e e n  elongated  manchette  47.  testis.  spermatid  - the p e r i p h e r a l 46.  o f t h e s p e r m a t i d s and  acrosomal granule  chromatin  Figure  sections  (NR), m a n c h e t t e which  have  (MA)  I t has and  increased  the  showing the dense  in size. '  12,000X. Figure  49.  The  nucleus  spermatozoon  (N) composed  showing  elongated  of a condensed  homogenous e l e c -  t r o n d e n s e m a t e r i a l w h i c h c o n t a i n s numerous v a c u o l e s ( V ) . The  cell  and  the n u c l e a r  30,000X.  membrane  (CM)  ring  appose,  (NR)  to the  is clearly  acrosome visible.  116  117 .Figures  50 t o 54.  separation head  B i z a r r e forms o f t h e acrosome  o f the acrosome  in testicular  from  and t h e  the nucleus of the  and c a p u t s p e r m a t o z o a  (sagittal  sections). Figure  50.  P y r i f o r m - s h a p e d acrosome  different testicular is  distribution  of acrosomal content  spermatozoon.  mostly apposed  (AC) s h o w i n g t h e  The c e l l  in a  membrane  (CM)  t o t h e a c r o s o m a l membrane.  45,000X. Figure  51.  The  Hook-shaped  acrosome  nuclear material  i n a caput  has a g r a n u l a r  spermatozoon.  appearance.  30,000X. Figure  52.  S-shaped  spermatozoon. (V). Figure  53.  acrosome  in a  testicular  The n u c l e u s h a s numerous  vacuoles  18,000X.' Knob-shaped  acrosome  i n a caput  spermatozoon.  30,000X. Figure  54.  The t e s t i c u l a r  s p a c e between  spermatozoon  t h e acrosome  However, t h e c e l l  membrane  to  25,000X.  t h e acrosome.  showing  distinct  (AC) and t h e n u c l e u s ( N ) . (CM) i s c l o s e l y  apposed  118  119 Figures  55 t o 58.  The movement o f t h e c y t o p l a s m i c  the  adhesiveness  the  acrosome,  of the c e l l  membrane  and t h e o c c u r r e n c e  droplet,  at the t i p of  of the s a t e l l i t e  fibrils. Figure  55.  Damaged and s e p a r a t e d  except  the t i p o f the acrosome  spermatozoon Figure  56.  caput (SF, Figure  Cross  (sagittal  spermatozoon arrows).  showing  intact  15,000X.  many  piece  satellite  s e c t i o n of a caput perforatorium  postacrosomal  plasmic  (AE) i s s e e n i n a c a p u t  section).  showing  (CM)  of a fibrils  80,000X.  Sagittal  acrosomal  membrane  s e c t i o n of the middle  57.  and  cell  content  droplet  swellings  spermatozoon  (P), acrosomal  (AS)  (PS) e v e n  though the  has been d i s l o d g e d .  The c y t o -  (CD) r e m a i n s  on t h e neck  region.  24,000X. Figure  58.  Axial  spermatozoon on the  the d i s t a l  s e c t i o n of the t a i l showing  plasmic 12,000X.  the c y t o p l a s m i c  end o f t h e m i d d l e  lower r i g h t droplet  of a cauda  side, a cross and t h e m i d d l e  piece  droplet (MP).  (CD) On  s e c t i o n of the cytopiece  i s shown.  120  121 E.  DISCUSSION Spermatozoa  transported deferens the  from  before  the  the  As immature Bedford tion  from  the e l e c t r o n  the  the  of  protoplasmic  the c o n t r a r y , Fawcett acrosomal  regions  micrographs  of  duct  the  i n the  rabbit,  epididyrnal  duct  of  occurrence  of  immature  spermatozoa  epididymis  as  observed  in this  mixing for  takes  the r a b b i t  considerable epididymis 1.  place within  The  The  and  pig.  mixing  of  the  Viability  motility  the epididyrnal  of  through  the  and  in this  no  the  epididymis.  absolute  with  any  rabbit.  correla-  particular On  spermatozoan  examining  the  guinea  with  suggests  epididymis Smidt  that  On  that  particular  pig.  i n a l l parts of  The the spermatozoan  i n t h e mink  as  reported  (1976) r e p o r t e d  spermatozoa c o u l d occur  also  neck.  (1963) i n d i c a t e d  correlated  study  H o l t z and  study,  O r g e b i n - C r i s t (1967a)  Hollenberg  c h a n g e s were c l o s e l y  the  vas  passage  i n a l l p a r t s of  d r o p l e t i n the and  and  are  eggs.  of maturation  epididyrnal  cauda e p i d i d y m i s  their  t h a t t h e r e was  stages  tubules  s p e r m a t o z o a become m a t u r e  to f e r t i l i z e  (1965b) r e p o r t e d  segment o f  the  During  s p e r m a t o z o a were f o u n d  of d i f f e r e n t  found  the  seminiferous  v i a the e p i d i d y m i s  ejaculation.  ability  seen  i n the  testis  reproductive tract,  acquire  the  produced  within  that  the  pig. of  Spermatozoa  of epididyrnal  duct  was  spermatozoa r e l e a s e d  significantly  i n c r e a s e d from  from the  caput,  122 via  the c o r p u s , t o cauda  epididymis.  s p e r m a t o z o a were n o t m o t i l e s p e r m a t o z o a was motility  was  (Gaddum,  1968),  ever,  than that  of cauda  ram and b u l l  (Setchell  e t a l . , 1969).  s p e r m a t o z o a o f t h e ram  (Voglmayr  1974),  and a l s o  F o o t e , 1968; Amann and A l m q u i s t , 1975 ),  of m o t i l i t y  and human  of t e s t i c u l a r  reports  and G l o v e r ,  (ElWishy,  1975),  1974), The  through  1967a),  bull  (Amann  and human  increase  mouse  et a l . ,  o f t h e mink was  mink  than t h a t  stallion  reported  The m o t i l i t y  (84.0% i n t h i s  higher  However,  rapid  passage than  that  increase i n  i n the b u l l  (Amann  spermatozoa  and i n the  s t u d y and 8 1 % by Ahmad e t a l . , 1975b) was  of the b u l l  (10-40%;  during  1975) o r r a b b i t  a rapid  of cauda  ass, s t a l l i o n  1973).  more  and F o o t e , 1 9 6 8 ) .  1962b).  of  1962b; Amann and  motility  (Paufler  Almquist,  1973).  low  motility  (Hoppe, 1 9 7 5 ) ,  and A l m q u i s t ,  (Bedford  was  et a l . ,  study are observations  f o r the s t a l l i o n , ass (ElWishy,  motility  1962b),  spermatozoa,  reported  spermatozoan  of the  and F o o t e , 1968; Gaddum,  of spermatozoan  the epididymis  (Amann  on s p e r m a t o z o a f r o m t h e r a b b i t  1965; P a u f l e r  Orgebin-Crist,  Griel,  (Bedford  o f c a p u t s p e r m a t o z o a , and i n c r e a s e d  agreement w i t h  1968;  1968) and b u l l  i n the caput spermatozoa  s p e r m a t o z o a as o b s e r v e d i n t h i s  (Gaddum  How-  i n the t e s t i c u l a r  absence  cauda  Slight  was  (Igboeliand  motility  spermatozoa.  weak movement a t b e s t ,  s t a l l i o n , ass (ElWishy, The  of e j a c u l a t e d  or only  Griel,  bull  testicular  spermatozoa of the r a b b i t  a l . , 1966, 1967; M u r d o c h and W h i t e ,  and  in  and t h e m o t i l i t y  observed i n t e s t i c u l a r  no m o t i l i t y ,  detected et  less  However,  ( 3 2 . 1 % ; Amann and A l m q u i s t ,  E l W i s h y , 1975),  ass (10-50%;  1962b),  E l W i s h y , 1975 )  123 and  rabbit  (61%; Paufler  estimation mink The  of m o t i l i t y  spermatozoa striking  through  and F o o t e , 1 9 6 8 ) .  i s somewhat s u b j e c t i v e ,  i s definitely  increase  higher  the reproductive acquire  which might  be r e l a t e d  tract  high  motility  o f t h e mink  motility  within  to the short  the value f o r  than f o r o t h e r  o f spermatozoan  spermatozoa  Although the  species.  during  implies  a short  passage  that  time  mink  period,  b r e e d i n g season of the  mink. The  motility  compared  to that  of e j a c u l a t e d o f cauda  between t h e e j a c u l a t i o n influence ductive  In s p i t e spermatozoan percentage trend.  and m o t i l i t y  of increasing  1962a;  compared  spermatozoa of  dead  spermatozoa change  be a r e s u l t  permeability  or the  female  repro-  Igboeli  spermatozoa  tract,  t h e mean  from  studies  specific  i n the  1965) and b u l l  and F o o t e , 1 9 6 8 ) .  A  i n t h e c a p u t and c o r p u s  to the t e s t i s  where t h e m o t i l i t y o f a s an  e_t al_. , 1942; G l o v e r ,  i n the s t a i n i n g  of a temporary  d u r i n g the  d i d n o t show a n y  Gaddum and G l o v e r ,  (Lasley  the time  motility  was a b s e n t , c a n n o t be i n t e r p r e t e d  However, t h i s might  spermatozoan  agrees with the r e s u l t s  of unstained  epididymis  estimation  lower  factors.  spermatozoa  (Amann and A l m q u i s t , 1962b; decrease  Perhaps  through the r e p r o d u c t i v e  of unstained  (Glover,  was  o f t h e male a n d / o r  are responsible  transit  This  rabbit  spermatozoa.  of the s e c r e t i o n  tract  spermatozoa  1962b).  b e h a v i o r of spermatozoa  increase  a s s u g g e s t e d by Amann  increase  i n membrane  and A l m q u i s t  (1962b).  124 2.  Changes  i n the Spermatozoan  Head  The  total  head  different  parts  of the r e p r o d u c t i v e  cally  different.  acrosomal cantly the  greater  pass  and  tract  the a n t e r i o r  of t e s t i c u l a r  spermatozoa  t h i c k n e s s o f mink  along the r e p r o d u c t i v e  tract  (Fawcett  Phillips,  1971),  stallion,  ram,  However, no human  hare,  modification  acrosome  a p p a r a t u s and through  actual  The vacuoles coupled  of  rats  and  i n volume, o r  c o n t e n t s has  mice  tract. just  with  the  between  as  they  with  boar  ( B e d f o r d and  (Jones,  1971),  r a t (Bedford,  (Bedford,  and  shape  Whether  in  1975). Golgi as  spermatozoa  t h e r e i s an  a redistribution  size  1975).  (Jones,  of  1971).  of the n u c l e a r  t h e n u c l e u s and  the  acrosome,  i n c r e a s e d e l e c t r o n d e n s i t y o f t h e a c r o s o m e r e v e a l e d by of spermatozoa  Kihlstrom  p a s s i n g down t h e e p i d i d y m i s  specific  to the d e h y d r a t i o n of and  monkey  not been d e t e r m i n e d  i n the space  of  acrosomal  p r o d u c t o f the  d e c r e a s e i n t h e number and and  three parts  pig, chinchilla  tailed  in size  the r e p r o d u c t i v e  micrographs  Lindahl  white  and  signifi-  t h e a c r o s o m e v/as r e p o r t e d  greatly  i n d i c a t e an i n c r e a s e i n t h e due  and  i s a secreted  varies  reduction  acrosomal  the  dog,  rabbit,  ( D i c k e y , 1965),  ( B e d f o r d , 1973), The  pass  bull  1969a),  length  were  i s i n agreement  i n the c a s e s o f the g u i n e a  Nicander,  from  from  statisti-  spermatozoa  observations and  were n o t  T h i s d e c r e a s e i n the a n t e r i o r  acrosomal  taken  acrosomal  than those of spermatozoa  epididymis.  length  l e n g t h s of the spermatozoa  However,  thickness  Length  gravity  spermatozoa  (1952).  as  of  spermatozoa  suggested  by  may  125 While change, during Since with  the e q u a t o r i a l  length  the postacrosomal length the spermatozoan  passage  t h e ovum, t h i s  increased  to the t o t a l  through  the r e p r o d u c t i v e  significantly testis  acrosomal The  tract.  since  of ejaculated  an  Changes  artifact  might  i n the Morphology  abnormal  This  a s s and  length  anterior  of the  latter.  than those tract.  decrease i s not c l e a r , of preparing  from  cauda  from the r e p r o d u c t i v e  i t may  samples  o f t h e male and/or  of the Spermatozoan  a c r o s o m e o f mink  be or  female  i s i n agreement  t h a n i n t h e c o r p u s and with  and F o o t e , 1 9 6 8 ) , b u l l stallion  (ElWishy,  epididymis.  Head  s p e r m a t o z o a were o b s e r v e d  results  obtained  (Amann and  1975).  spermatozoa decreased during the  the a p i c a l  the d e c r e a s e . of the  and c a p u t e p i d i d y m i s  (Paufler  1962b),  passage  t o t h e c o r p u s and  of the process  of  tract.  the t e s t i s  epididymis.  length  the passage of spermatozoa  of the s e c r e t i o n  More a b n o r m a l  through  tract.  contact  s p e r m a t o z o a were s h o r t e r  the reason f o r t h i s  an i n f l u e n c e  rabbit  of  t h e f o r m e r i s a component  the spermatozoa c o l l e c t e d  by  during  However,  caput epididymis  length  reproductive  in  site  the nuclear  significantly  i n accordance with  head  Although  3.  through the r e p r o d u c t i v e  length,  decreased during  and  epididymis,  by  Increased  postacrosomal length  head  spermatozoa d i d not change  caused  definite  fertlization.  Similar  of  of spermatozoa  the p o s t a c r o s o m a l sheath i s the f i r s t  enhance  the  d i d n o t show any  The  cauda  i n the  Almquist,  ratio  of  the spermatozoan  passage  126 The the  cell  acrosome  However,  membrane o f s p e r m a t o z o a as s p e r m a t o z o a  t h e r e was  no  significant  spermatozoa  the  tract.  found  loosely  ejaculated due  fitted  rabbit  partially  Zamboni and several mal  kinds  less,  their  report  the r a b b i t .  glutaraldehyde  cacodylate.  in  mM  acrosome  solution  cell  from  spermatozoan  passage  150  mM  the  separating  tozoa  sodium  also  e_t _al_.  fixed  Jones  mM  mM  o f the c e l l  might  of  be  peri-  compared 150  with  mM  was  got  in this  study,  during  tract  even  used.  the though  Therefore,  the acrosome a  fixed  the  solution  t h e acrosome  membrane f r o m  p a s s down t h e e p i d i d y m i s  Neverthe-  spermatozoa  However,  fixative  cell  spermatozoa  apposition  structure.  buffered  as  sperma-  species-specific  phenomenon. As  shown i n e l e c t r o n  spermatids testis. from  has been  micrographs, microstructure  o b s e r v e d i n sperm  S p e r m a t i d s have  also  the c a p u t e p i d i d y m i s .  been  be  epididy-  of the  and  through the r e p r o d u c t i v e  cacodylate  might  spermatozoa.  (1971)  100  i n 100  from  in  compared  and mouse  configurations  in close  separating  (1968)  preservation.  with  of  Furthermore,  concluded that  that  this  c a p u t and c a u d a  buffered  the s u b j a c e n t  membrane was  the acrosome  ejaculated  spermatozoa,  remained  membrane w h i l e  separated the  She  (1963b)  configuration  showed d i f f e r e n t  membranes between  sodium 150  rabbit  of poor c e l l  fixatives  and Hadek  used f o r r a b b i t  the abnormal  In boar  parts  procedure.  human and  the r e s u l t  acrosomal c e l l of  and  i n t h e damaged  suggested that  ( 1 9 6 8 ) , Zamboni  of f i x a t i v e s  concluded that  membrane was  and  tract.  successive  membrane a r o u n d  t o the f i x a t i o n  spermatozoa  They  taken from  spermatozoa  Stefanini  difference  B e d f o r d (1964)  cell  from  p a s s dov/n t h e r e p r o d u c t i v e  membrane b e t w e e n reproductive  tends to separate  samples  of  taken from the  o b s e r v e d i n samples  Although the epididymis  taken  is a  127 spermatozoan caput is  transport,  e p i d i d y m i s might  directly  (Clermont  connected  and  than caput explain  spermatozoa  Except  swellings mink  on  moved  head  had  was  and  seen  contact  tract.  fertility  to  the  reproductive  the o c c u r r e n c e of  between  as  Movements o f t h e C y t o p l a s m i c  occurred  during  and  the caput  incidence  of  spermatozoa  change might  the e n t e r i n g segment  be  because  spermatozoon or  post-  Droplet  d e c r e a s e " i n the c y t o p l a s m i c d r o p l e t  spermatozoan  transit  from  from  the t e s t i s  to corpus e p i d i d y m i s .  the c y t o p l a s m i c d r o p l e t  Although  the c o r p u s , cauda Branton  and  was  T h e r e f o r e , the  corpus  to cauda that  such  low  recorded i n  e p i d i d y m i s and  Salisbury  to caput  ejaculated  (1947) r e p o r t e d  d r a m a t i c d e c r e a s e of the p r o x i m a l c y t o p l a s m i c d r o p l e t  indicated  the  region.  A significant  the  the  spermatozoa  of spermatozoa  t h e mammalian ovum i s t h e e q u a t o r i a l  semen.  to  of the  This  and  from  swellings  compared  to i n c r e a s e  site  4.  testis  p o s t a c r o s o m a l segments of  the  acrosomal  of the  an  ratio  case, generally,  the r e p r o d u c t i v e  initial  difficult  the other p a r t s  t o t h e m a t u r i t y and of  is  swelling  the e q u a t o r i a l  through  related  from  for this  spermatozoa  the e p i d i d y m i s  more a c r o s o m a l  spermatozoa  acrosomal  removed  had  This  caput  low  efferentes  the  1961).  spermatozoa  because  storage organ,  spermatids because  with d u c t u l i  spermatozoa.  inordinately  tract.  have  Huckins,  Testicular  m a t u r a t i o n and  e p i d i d y m i s of the b u l l , a d e c r e a s e o c c u r r e d as  many  a from  investigators  spermatozoa  passed  128 from  the c a p u t  to the  corpus  (Bedford,  1965b; O r g e b i n - C r i s t , 1967a; P a u f l e r and (1957b) a l s o r e p o r t e d  away f r o m  the  neck  spermatozoa,  from  the  distal  with  the  results  It  caput  Orgebin-Crist the  cytoplasmic  caput of  to corpus  the  is interesting  cytoplasmic  to note  s p e r m a t o z o a was  i n mink  (Tables  detachment  of  associated  with  concluded,  for instance,  not  the  rabbit.  5.  Changes  protoplasmic  i n the  activities  were f o u n d  The tion the  chemical  testicular  and and  the  finding  agrees  study. that while  t o be  the r a t i o  strikingly the  the  of  indicate  from  of  these  to  the  that  the  corpus  spermatozoa Bedford  the m o t i l i t y  the  reduced  motility  of c y t o p l a s m i c  is  cauda  simply  (1967b)  of caput  spermatozoa  droplets in  Activity  the  reproductive  d r a m a t i c a l l y reduced  composition  (Roussel  This  tract,  most  s p e r m a t o z o a were e n h a n c e d , w h i l e  s p e r m a t o z o a when compared  of  occurred  presence  through  of  end  droplet  droplet migration  d r o p l e t s from  Enzyme  passage  enzyme a c t i v i t i e s  the  the  1968).  that, for  T h i s may  that  rabbit  Foote,  proximal  spermatozoan m a t u r a t i o n .  i n f l u e n c e d by  During  4).  and  and  (1967a) c o n c l u d e d  spermatozoa,  2 and  (Amann  movement o f  i n the  g r e a t l y i n c r e a s e d from  epididymis  was  found  the  1975),  epididymis.'  present  d r o p l e t was  to corpus  that  always o c c u r r e d  epididymis.  most  (ElWishy,  bull  1962b),  corpus  stallion  i n the  Almquist,  Nicander  ass,  epididymis  with  those  (White,  Stallcup,  1966;  from  those  in ejaculated the  1973a) and  cauda  enzyme  epididymis. distribu-  E i n a r s s o n e t a l . , 1976)  epididyrnal f l u i d  are  different.  of  Therefore,  129 the d i f f e r e n c e environmental along in  i n t h e enzyme r e a c t i o n c h a n g e e n c o u n t e r e d by  the r e p r o d u c t i v e  tract  On  the other  (1975) a l s o  from  phosphatase  extent  acid  spermatozoa  from  the ductus d e f e r e n s .  mink  spermatozoa  reproductive swellings,  as  during  tract  may  The  phosphatase  spermatozoa  Glover,  Einarsson  phosphatase  increased  feature  epididymal f l u i d  i n the  i t was  to a  absent in  through the  spermatozoa.  spermatozoa  the r e p r o d u c t i v e  phosphatase  also tract.  activity  (Bavdek  e_t a l . (1976 ) r e p o r t e d  the  the t e s t i c u l a r  i n the b o a r .  head  to the occurrence  spermatozoa  from  Poirier  c a p u t , and  transit  i n mink  alkaline  activity  that  enzyme a c t i v i t y  i n mink  through  of rabbit  but  in addition  activity  moved  droplet  1970).  epididymis,  indicate,  the c y t o p l a s m i c  the cauda  increase  reported  activity  the t e s t i s ,  the spermatozoan  agrees with i n c r e a s i n g  alkaline  from  another s p e c i a l  Alkaline  of  pass  o f t h e enzyme  epididymis,  phosphatase  the cauda  from  This  they  i n r a t spermatozoa decreased  the c a p u t to cauda  demonstrated  membrane o f m i c e  increased  as  to the  ( W h i t e , 1973b) o r t o an  hand, T e r n e r e_t a l . ( 1975)  of a c i d  transit  smaller  due  1970).  the a c t i v i t y during  be  spermatozoa  t h e amount o f enzyme, o r an u n m a s k i n g  (Voglmayr,  of  might  However,  the  o f t h e enzyme i n mouse s p e r m a t o z o a d e c r e a s e d d u r i n g t h e i r  in  and increase fluid  to  activity transit  t h r o u g h t h e e p i d i d y m i s ( T e r n e r et. a l . , 1975) and i n t h e c y t o p l a s m i c droplet  of rabbit  Moniem and  spermatozoa  Glover,  G l o v e r (1972) s u g g e s t e d t h e d i f f i c u l t y  c h e m i c a l m e t h o d s and as p o s s i b l e  (Moniem and  the s u b j e c t i v i t y  o f some  reasons f o r the c o n t r a d i c t o r y  1972). of  histo-  interpretations  results.  130 Voglmayr ejaculated ram.  et. _ a l .  (1969)  spermatozoa  However, Gordon  periacrosomal  reported  than i n t e s t i c u l a r (1973)  portion  recorded  from  the cauda  spermatozoa  showed no r e a c t i o n .  ejaculated  mink  in  the r a b b i t  and  activity than  was  spermatozoa human.  slightly  ATPase  activity  and MDH  of t h i s  this  might  ejaculated that  and  1971)  completely inhibits  and  Spermatozoa  also  study.  that  (Lardy  there  rabbit  and  of e i t h e r  activities  of  Phillips, inhibitor  several of  reported  activity  dehydrogenase and  i n t h e semen iodoacetic  and  no p h o s p h a t a s e  from seminal  human s p e r m a t o z o a .  In t h i s  Gordon  activity  G-^6-Pase and  washed o r unwashed  alkaline  1941c).  ( Z a n e v e l d , e t a_l. , 1 9 7 3 ) . was  reactions  spermatozoa i n  mating,  low c o n c e n t r a t i o n  ADPase, a d e n o s i n 5 *-monophosphatase, t h e plasmalemma  which  epididymis  I t has been  phosphatase  absorb a proteinase  ejaculation  (1973) r e p o r t e d  cauda  p h o s p h a t a s e s , ADPase,  the e j a c u l a t e d  succinate  i n spermatozoa  during  the  spermatozoa.  the cauda  some enzyme  acid  phosphatase  plasma  of  found  dehydrogenase  from  from females a f t e r  in this  inhibits  Bami,  Since  inhibited  spermatozoa  L-tartrate  (Sivaram acid  spermatozoa.  have  with that  e t a_l. , 1975 ),  alkaline  human  activity  d e c r e a s e d t o weak o r n e g a t i v e  s t u d y were c o l l e c t e d  factors  and  and  o f ATPase  s t u d y I n mink  spermatozoa  were f o u n d d r a m a t i c a l l y ejaculated  (Terner  i n the  ejaculated  absence  i n spermatozoa  of a c i d  i n mink  while  i s i n agreement  from the c a p u t e p i d i d y m i s  Strong  The  of r a b b i t  in  of the  activity  In the r a t , l a c t a t e  higher  agrees with the r e s u l t s  in  epididymis,  activity  spermatozoa  ATPase  o f t h e plasmalemma  spermatozoa  in  more ATPase  (ATPase,  others) in  ejaculated  s t u d y unwashed  131 ejaculated but  s p e r m a t o z o a were e x a m i n e d  here a l s o  the  results  A significant epididymal seyeral  decrease  mammals  (Zaneveld  premature  in  reproductive  female  activity al.,  increased  1969b).  involved  and  these  spermatozoan tract.  of  acid  and  A T P a s e , w h i c h m i g h t be  The  from  significant  spermatozoa  from  Afterv/ards, the  (Zaneveld  transport proteinase  (Zaneveld  remove any  et.  inhibitor  e_t _al_. ,  1972).  events,  i t i s assumed  that  alkaline  phosphatases,  ADPase  important during  in fertilization,  the  would  t r a n s p o r t a t i o n from  study  the  caput  i n d i c a t e d t h a t most  the  took  place  i n two  morphological  steps:  the  first,  testis  to the  caput  to  corpus  epididymis.  the  epididymis,  d i f f e r e n c e i n the m o r p h o l o g i c a l  from  inhibition  changes d u r i n g  may  in  uterus.  from the  head  inhibited  spermatozoa  passage  second,  or  the  present  changes of their  to  Such  i n c a p a c i t a t e d spermatozoa  successive  have been b l o c k e d  no  e_t a J . , 1973 ) .  spermatozoan  activity  •epididymis  negative.  acrosin activity  C a p a c i t a t i o n _in u t e r o  i n the  Considering strong  of  were  t o e j a c u l a t e d s p e r m a t o z o a v/as d e m o n s t r a t e d  v/ould p r e v e n t the  obtained  f o r enzyme a c t i v i t i e s  corpus  to the  cauda  during and  There  aspect  epididymis.  of  was  132 F.  SUMMARY The  m a t u r a t i o n changes  enzyme a c t i v i t y with  were s t u d i e d  spermatozoa  parts  in viability,  collected  of the epididymis  morphology,  i n the present  and  investigation  from the t e s t i s ,  three  and f r o m e j a c u l a t e d  successive  semen i n t h e  mink. The  motility  increased  (p < 0.05) d u r i n g  successive  parts  spermatozoa compared  spermatozoa  the  was  was  changed  very  ficantly  of cauda  spermatozoa.  Although the t o t a l  little,  the a n t e r i o r  While  head  less  active  testicular  high  per cent of  their  length  transit in  of  spermatozoa  d e c r e a s e d (p < 0.001) w h i l e t h e p o s t a c r o s o m a l  length  significantly  the a p i c a l  though  smaller  epididymis  than that  moved  significantly from  maintained almost s i m i l a r  significantly  tract.  decreased  epididymis.  The head  smaller  those of spermatozoa  than  tract.  during  obtained  (p <  the nuclear passage of  the t e s t i s  of ejaculated  testicular  spermatozoa.  thickness  (p < 0.001) f r o m  of  and c a p u t  epididymis,  values  from the  decreased  the t e s t i s  The a c r o s o m a l  length  length  of epididyrnal  t o t h e c o r p u s and c a u d a  reproductive  transit  The e q u a t o r i a l  length  0.001) when s p e r m a t o z o a  acrosomal length  (p < 0.05) d u r i n g  to the epididymis.  Even  tive  Ejaculated  m a t i n g was  during  through  signi-  was  the  females a f t e r  by s p e r m a t o z o a  passage  system.  non-motile, a comparatively  spermatozoa  length  the spermatozoan  significantly  was  increased testis  from  acquired  epididymis.  was f o u n d t o be  of the reproductive  collected  to that  motility  of spermatozoa  through  spermatozoa to the  s p e r m a t o z o a were from  the reproduc-  133 During matrix and  transit  appeared  size  tract,  t o become more e l e c t r o n  dense;  of nuclear  spermatozoa T h e r e was the  i n the r e p r o d u c t i v e  vacuoles decreased.  were f o u n d  an  acrosome  increase  i n the t e s t i s of separation  (p < 0.05)  as  tract.  a c r o s o m e was  more p r o n o u n c e d  was  no  statistical  from d i f f e r e n t of  Such  cell  swelling  significantly  and  caput spermatozoa  all  ejaculated The  incidence  p a s s e d down  had  spermatozoa  the from  the There  removed  i n t h e damage  from  testicular  spermatozoa.  Almost  swellings. droplet  testicular  A few  tract  cauda  of the c y t o p l a s m i c  spermatozoa.  from  spermatozoa.  (p < 0.01)  t o c o r p u s and  from  membrane  o c c u r r e n c e of the p o s t a c r o s o m a l  increased  (p < 0.001) d i m i n i s h e d epididymal  The  spermatozoa  epididymis.  membrane s e p a r a t i o n  between  number  abnormal  of the c e l l  of the r e p r o d u c t i v e  t h e a c r o s o m a l membrane.  the  caput  in ejaculated  difference  parts  and  spermatozoa  reproductive  Many  the acrosomal  significantly  spermatozoa  ejaculated  to  spermatozoa  had  the  droplet. The  a c t i v i t i e s of several  were i n c r e a s e d  during  enzymes, e x c e p t DOPA  the spermatozoan  reproductive  tract.  and  p h o s p h a t a s e s , ADPase, A T P a s e and  alkaline  dehydrogenase) from  However,  transit  were f o u n d  f e m a l e mink  after  through  l o w e r e d enzyme a c t i v i t i e s  in ejaculated  mating.  oxidase, the (acid  malate  spermatozoa  recovered  GENERAL The  head  flattened change  and  ovate  the  s p e r m a t o z o a was  in outline.  significantly  through and  o f mink  during  testis,  cauda).  SUMMARY  the  and  However,  The the  total  passage of  epididymis the  head  s p e r m a t o z o a were s m a l l e r t h a n reproductive  The  dorsoventrally  anterior  of  t w o - t h i r d of be  divided  into  covered  with  spermatozoan  transit  through  main  (p <  acrosomal  segments of  0.001), w h i l e  the  the  a c r o s o m e , was  (p <  0.05).  decreased  (p <  0.001) d u r i n g  the  and  caput  acrosomal  during  with  t o be  vacuoles  i n the  and  the  disappeared.  decreased  sheath.  the  tract, apical  decreased  significantly significantly  cauda  from  epididymis;  In  the acrosomal  t h e number  spermatozoa  the  (p •£ 0.001)  epididymis.  and as  and  The  and  matrix  size  passed  of  down  tract.  intact On  dense  of  spermatozoa  i n dimensions,  more e l e c t r o n nucleus  composed  decreased  to the  content.  significantly  of  (apical,  the r e p r o d u c t i v e  length also  transit  testis  p e r f o r a t o r i u m was  remained  the  with  postacrosomal  to the c o r p u s  the changes  reproductive The  apical  epididymis  from  appeared  the  The  covered  l e n g t h was  thickness s i g n i f i c a n t l y  transit  accordance  the  postacrosomal  was  acrosomal  l e n g t h , w h i c h was  increased  testis  from  t h r e e segments  o n e - t h i r d was  and  collected  the n u c l e u s  posterior  anterior  corpus  ejaculated  spermatozoa  e q u a t o r i a l ) a c c o r d i n g to the  the  not  spermatozoa  (caput,  m a i n , and  the  length did  tract.  acrosome which can  During  head  even  triangular  though  the d o r s a l  and  the  in longitudinal  acrosomal  ventral  sections  content  aspects of  the  head,  135 two  s w e l l i n g s as  anterior  border  swelling  was  sheath.  A  these  hump-like  of the e q u a t o r i a l  also  found  strong acid  during  the  c a u d a and  composed In  of  ejaculated  of  separating  from  passed  the c e l l  remained  intact.  The  and  laterally  and  proper,  tail  except  activity  and  i n the  five  minor columns, The  caput  Almost a l l  sheath  and  was  membrane.  at the  to  be  t i p , as  (p <  0.05).  was  not  localized  s w e l l i n g s but  also in  spermatozoa.  Non-  postacrosomal  sheath.  phosphatase,  ADPase,  d e h y d r o g e n a s e were a l s o  localized  spermatozoa. of  the d o r s o v e n t r a l l y  c o l u m n s w h i c h was the  0.01)  sheath  of a l k a l i n e  malate  and  of  postacrosomal  of epididymal  localized  at  membrane a p p e a r e d  separated capitulum connected  striated  occurrence  the c e l l  postacrosomal  composed  of  scrolls.  r e g i o n the c e l l  of epididymal  n e c k was  The  testis  postacrosomal  d e p o s i t e d on  the a c t i v i t i e s  ring  and  The  phosphatase  A T P a s e , DOPA o x i d a s e t h e head  located  the acrosomal  membrane c o v e r i n g t h e  e s t e r a s e was these,  was  i n c r e a s e d (p <  the  showed  the  postacrosomal  activity  from  at  similar  cauda e p i d i d y m i s .  acrosomal  acrosomal  the g a l e a c a p i t i s  Besides  the  down t h e r e p r o d u c t i v e t r a c t  A strong acid  specific  and  layer  the  However,  i n the  of  One  spermatozoa.  spermatozoa  acrosomal  spermatozoa  side  spermatozoa  swellings.  a dense  the  segment.  phosphatase  to the corpus  postacrosomal  but  each  were f o u n d  s w e l l i n g s v/as s i g n i f i c a n t l y  passage  epididymis  in  on  swellings i n epididymal  postacrosomal  only  structures  remnants of  p r o x i m a l l y with  f o l l o w e d by  proximal c e n t r i o l e ,  continuous  two  a few  major  a  and  mitochondria  t h e axoneme were f o u n d  i n the  136 interior  o f t h e column  The  tail  bundle.  consisted  o f n i n e dense  fibers,  microtubules,  and two c e n t r a l  fibers  9, 1, 5 and 6 were l a r g e r  dense was  number fibers.  larger  piece,  than  the s u b f i b e r fiber  coiled  a n n u l u s was  piece  from  testicular  from  corpus,  cauda  caput  and e j a c u l a t e d  motility  < 0.05) f r o m  motile  decreased  spermatozoa. spermatozoa  fibrous had  aspects.  The  (p <  0.001)  Within the was f o u n d  markedly  T h e r e f o r e , a few had t h e d r o p l e t  on  piece. was  significantly  spermatozoa  i n the cauda  spermatozoa  The  of the t a i l ,  spermatozoa.  of spermatozoa  no m o t i l e  spermatozoa  ejaculated  sections.  of the  The o c c u r r e n c e o f t h e  significantly  to corpus  of the middle  The shape  piece  pattern.  to epididyrnal  of the  of s u b f i b e r A  a l o n g the d o r s o v e n t r a l  c a u d a l end o f the m i d d l e The  (p  was  dense  s u r r o u n d e d by 53 t o 57  the occurrence of the d r o p l e t  reduced  the  was  i n longitudinal  the 9 + 2  droplet  epididymis,  bundle  columns  retained  cytoplasmic  the r e s t  In t h e r e g i o n  covered the p r i n c i p a l  two l o n g i t u d i n a l end  than  gyres of mitochondria.  triangular  sheath, which  B.  double  The d i a m e t e r s o f  In the d o u b l e t s , the diameter  the a x i a l  helically  fibers.  nine  collected  i n the t e s t i s  epididymis. from  female  increased t o 84%  The m o t i l i t y o f after  coitus  was  6 5%. A number  o f enzyme  6-phosphogluconic epididyrnal lactate  dehydrogenase,  spermatozoa.  and i s o c i t r a t e  were c o n f i n e d  activities,  with  the e x c e p t i o n of  were l o c a l i z e d  Glucose-6-phosphatase, dehydrogenases,  t o the middle  piece,  i n the t a i l  of  succinate,  and NADH d i a p h o r a s e  w h i l e malate  dehydrogenase  137 activity the  tail.  middle  The enzyme 5 - n u c l e o t i d a s e  activities  the e x c e p t i o n  during  b a s e and t h e p r i n c i p a l was  piece of  also confined  to the  piece.  The with  e x t e n d e d t o t h e head  passage  activities  o f t h e s e enzymes o f DOPA o x i d a s e ,  of acid  and a l k a l i n e  immediately  following  tract.  mating.  enhanced  However, t h e  p h o s p h a t a s e s , ADPase,  were s i g n i f i c a n t l y  spermatozoa which  spermatozoa,  were f o u n d t o be  through the r e p r o d u c t i v e  and m a l a t e d e h y d r o g e n a s e ejaculated  i n mink  were r e c o v e r e d  ATPase,  lowered i n f r o m f e m a l e minks  138 LITERATURE  CITED  A c k e r m a n , D.R. 1972. M i c r o s p e c t r o p h o t o m e t r y o f t h e DOPA r e a c t i o n i n human s p e r m a t o z o a . F e r t i l . S t e r i l . 23 : 4 3 - 4 9 . Adams, C . E . 1 9 7 5 . A r t i f i c i a l i n s e m i n a t i o n i n t h e mink, Mustela v i s o n . B r i t i s h Fur Breeders Gazette. 46 : 67-72. A f z e l i u s , B.A. 1 9 5 5 . 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