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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.Sc., Chung-Buk U n i v e r s i t y , Korea, 1956 M.Sc, Tohoku U n i v e r s i t y , Japan, 1970 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of ANIMAL SCIENCE We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA December, 1976 @ Jong-Wook Kim, 1976 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes i s for f i nanc ia l gain sha l l not be allowed without my wr i t ten permiss ion. Department of Animal Science The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date i i ABSTRACT T h i s study was undertaken to i n v e s t i g a t e the u l t r a -s t r u c t u r e , c y t o c h e m i s t r y and m a t u r a t i o n changes of mink spermatozoa which are important i n b i o l o g i c a l r e s e a r c h and i n t h e i r r e l e v a n c e to a r t i f i c i a l i n s e m i n a t i o n . Mature standard dark mink were used i n t h i s s t u d y . Spermatozoa were r e l e a s e d from the t e s t i s and e p i d i d y m i s (caput, corpus and cauda) of the male mink, and were a l s o c o l l e c t e d from the vagina of female mink immediately a f t e r mating. C o n v e n t i o n a l l y prepared t h i n s e c t i o n s were observed under a t r a n s m i s s i o n e l e c t r o n microscope. Enzymes were c y t o c h e m i c a l l y l o c a l i z e d i n spermatozoa. The mink spermatozoon head showed s i x s w e l l i n g s on the d o r s o v e n t r a l a s p e c t s : two connected hump-like s t r u c t u r e s at the a n t e r i o r border of the e q u a t o r i a l segment of the acrosome, and one at the postacrosomal sheath on each s i d e . These s w e l l i n g s , which show a s t r o n g a c i d phosphatase a c t i v i t y , appeared to be a s p e c i e s - s p e c i f i c s t r u c t u r a l f e a t u r e which might be n e c e s s a r y f o r the r e c o g n i t i o n o f the ovum or f o r sperm-ovum attachment i n f e r t i l i z a t i o n . The o c c u r r e n c e of the postacrosomal s w e l l i n g i n spermatozoa was s i g n i f i c a n t l y i n c r e a s e d (p < 0.01) d u r i n g the passage of spermatozoa through the r e p r o d u c t i v e t r a c t . Although the t o t a l l e n g t h of the head d i d not change s i g n i f i c a n t l y d u r i n g the passage of spermatozoa down the r e p r o d u c t i v e t r a c t , the a n t e r i o r acrosomal l e n g t h was s i g n i f i c a n t l y decreased (p < 0.001), w h i l e the postacrosomal l e n g t h was s i g n i f i c a n t l y i n c r e a s e d (p < 0.05). The c e l l membrane on the p e r i p h e r a l p a r t of the acrosome, w i t h the e x c e p t i o n of the t i p of the acrosome, was s i g n i f i c a n t l y s eparated (p < 0.05) d u r i n g the passage of spermatozoa through the r e p r o d u c t i v e t r a c t . The neck appeared to show d o r s o v e n t r a l l y c o n t i n u o u s but l a t e r a l l y s e p a r a t e d c a p i t u l u m which was f o l l o w e d by two major and f i v e minor columns, forming at f i r s t a s t r i a t e d r i n g and then j o i n i n g with the dense f i b e r s . o f the a x i a l f i b e r bundle. Some axoneme remnants were found i n the i n t e r i o r of the column bundle. The shape of the annulus was t r i a n g u l a r i n l o n g i t u d i n a l s e c t i o n s . The occurrence of the c y t o p l a s m i c d r o p l e t was s i g n i f i c a n t l y decreased (p < 0.001) d u r i n g the passage of spermatozoa through the 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 of ' spermatozoa was s i g n i f i c a n t l y i n c r e a s e d (p < 0.05) as spermatozoa passed the s u c c e s s i v e p a r t s of the r e p r o d u c t i v e t r a c t . The a c t i v i t i e s of a c i d and a l k a l i n e phosphatases, ADPase, ATPase and DOPA oxidase were found to be d i s t r i b u t e d i n the head, middle and p r i n c i p a l p i e c e s of ep i d i d y m a l spermatozoa. Glucose-6-phosphatase, 5 - n u c l e o t i d a s e , n o n - s p e c i f i c e s t e r a s e , malate, s u c c i n a t e , l a c t a t e and i s o c i t r a t e dehydrogenases, and NADH di a p h o r a s e a c t i v i t i e s were seen to be c o n f i n e d to the middle p i e c e , w h i l e the e s t e r a s e and malate dehydrogenase a c t i v i t i e s extended to the head base. The a c t i v i t y of 6-phosphogluconic dehydrogenase was not d e t e c t e d . Although most enzyme a c t i v i t i e s of spermatozoa were enhanced d u r i n g the passage of spermatozoa through the r e p r o d u c t i v e t r a c t , s e v e r a l enzyme a c t i v i t i e s ( a c i d and a l k a l i n e phosphatases, ADPase, ATPase, and malate dehydroge-nase) were d i s t i n c t l y reduced i n spermatozoa from e j a c u l a t e d semen r e c o v e r e d from female mink f o l l o w i n g mating. The presence of enzyme i n h i b i t i n g f a c t o r s i n the seminal plasma or female r e p r o d u c t i v e t r a c t was d i s c u s s e d . V TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS . v LIST OF TABLES i x LIST OF FIGURES x ACKNOWLEDGEMENTS x i GENERAL INTRODUCTION 1 CHAPTER I . AN ULTRASTRUCTURAL STUDY OF MINK SPERMATOZOA 3 A. I n t r o d u c t i o n 3 B. Review of r e l a t e d l i t e r a t u r e 4 1. E a r l y s t u d i e s of spermatozoa 4 2. General morphology of mink spermatozoa . . 5 3. The spermatozoan head 6 a. The nucleus 6 b. The acrosome 6 c. The p e r f o r a t o r i u m 8 d. The postacrosomal sheath 9 e. The c e l l membrane 9 4. The Spermatozoan neck 10 5. The spermatozoan t a i l 13 a. The a x i a l f i b e r bundle 13 b. The m i t o c h o n d r i a l sheath 16 c. The annulus (Jensen's r i n g , r i n g c e n t r i o l e ) 17 d. The c y t o p l a s m i c d r o p l e t 18 e. The f i b r o u s sheath 19 f . The end p i e c e ( t e r m i n a l p i e c e ) . . . . 19 C. M a t e r i a l s and methods 20 D. R e s u l t s 21 1. The spermatozoan head 21 2. The spermatozoan neck 23 v i Page 3. The spermatozoan t a i l 24 E. D i s c u s s i o n 36 1. The spermatozoan head 36 2. The spermatozoan neck 39 3. The spermatozoan t a i l 41 F. Summary 44 CHAPTER I I . CYTOCHEMICAL OBSERVATIONS OF MINK SPERMATOZOA 46 A. I n t r o d u c t i o n 46 B. Review of r e l a t e d l i t e r a t u r e 48 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 48 2. The r o l e of enzymes i n the spermatozoan m o t i l i t y 49 3. The r o l e of enzymes i n f e r t i l i z a t i o n . . . 51 4. Epididymal spermatozoa as a model f o r the enzyme study 53 Co M a t e r i a l s and methods 56 D. R e s u l t s 58 1. Phosphatases 58 2. E s t e r a s e and oxidase 58 3. Dehydrogenases 59 E. D i s c u s s i o n 63 1. Phosphatases 63 2. E s t e r a s e and oxidase 70 3. Dehydrogenases 72 F. Summary 78 CHAPTER I I I . ULTRASTRUCTURAL AND CYTOCHEMICAL CHANCES OF MINK SPERMATOZOA COLLECTED FROM THE REPRODUCTIVE TRACT AND SEMEN 80 A. I n t r o d u c t i o n 80 B. Review of r e l a t e d l i t e r a t u r e 82 1. E a r l y s t u d i e s of the r e p r o d u c t i v e t r a c t . . 82 2. M o r p h o l o g i c a l changes . 83 v i i Page a. The c y t o p l a s m i c d r o p l e t 83 b. The acrosome 84 c. The c e l l membrane 85 d. The middle p i e c e 87 e. Abnormal spermatozoa 87 f . S p e c i f i c g r a v i t y 87 g. R e f l e c t i n g c a p a c i t y to the l i g h t . . . 88 3. Chemical changes 88 a. Chemical c o m p o s i t i o n . . . . . . . . . 88 b. Enzyme d i s t r i b u t i o n 89 c. Metabolism 89 4. P h y s i o l o g i c a l changes 90 a. F e r t i l i t y 90 b. M o t i l i t y 92 c. E o s i n o p h i l i c p r o p e r t y 94 d. Temperature shock 94 e. R e s i s t a n c e to a l k a l i n i t y and a c i d i t y . 95 f . Net n e g a t i v e s u r f a c e charge 95 g. A g g l u t i n a t i n g p r o p e r t y 95 h. Contact a b i l i t y .96 C. M a t e r i a l s and methods 97 1. The p r e p a r a t i o n of m a t e r i a l from the r e p r o d u c t i v e t r a c t 97 2. The p r e p a r a t i o n of e j a c u l a t e d semen .and v i a b i l i t y t e s t 97 3. The procedure f o r e l e c t r o n microscopy . . . . 99 D. R e s u l t s . . 101 1. The v i a b i l i t y of spermatozoa 101 2. Changes i n the spermatozoan head l e n g t h . . 101 3. Changes i n the morphology of the spermatozoan head 106 a. The acrosome . 106 b. The c e l l membrane 107 c. Spermatism 107 d. The s w e l l i n g 109 v i i i Page 4. Changes i n the morphology of the spermatozoan t a i l 109 5. Changes i n the enzyme a c t i v i t y . . . . . . . 110 E. D i s c u s s i o n 121 1. The v i a b i l i t y of spermatozoa 121 2. Changes i n the spermatozoan head l e n g t h . . 124 3. Changes i n the morphology of the spermatozoan head 125 4. Movements of the c y t o p l a s m i c d r o p l e t . . . 127 5. Changes i n the enzyme a c t i v i t y 128 F. Summary 132 GENERAL SUMMARY 134 LITERATURE CITED 138 i x LIST OF TABLES No. T i t l e Page 1 The methods of enzyme l o c a l i z a t i o n 57 2 The per cent of m o t i l i l e spermatozoa 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 " the 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 3 Dimensional changes at d i f f e r e n t p o r t i o n s of the spermatozoan head 104 4 Changes i n the c e l l membrane c o n f i g u r a t i o n and i n the o c c u r r e n c e of s w e l l i n g s and the c y t o c h e m i c a l d r o p l e t 108 5 Changes i n enzyme a c t i v i t i e s l o c a l i z e d i n the spermatozoa from 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 LIST OF FIGURES No. T i t l e Page 1-4 U l t r a s t r u c t u r e of the epididyrnal spermatozoan head 28 5 - 8 U l t r a s t r u c t u r e of the spermatozoan neck . . . . 30 9-13 U l t r a s t r u c t u r e of the neck and middle p i e c e i n spermatozoa 32 14 - 23 U l t r a s t r u c t u r e of the spermatozoan t a i l . . . . 35 24 - 37 Enzyme l o c a l i z a t i o n i n epididyrnal spermatozoa 62 38 Schematic drawing of 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 of the v a r i o u s p a r t s (shaded r e g i o n s ) 98 39 The p o s i t i o n s of the head measured 100 40 - 44 Acrosomal changes of spermatozoan heads d u r i n g passage through the r e p r o d u c t i v e t r a c t and subsequent 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 45 - 49 S a g i t t a l s e c t i o n s of the spermatids and spermatozoa i n the t e s t i s 116 50 - 54 B i z a r r e forms of the acrosome and the s e p a r a t i o n of the acrosome from the nucleus of the head i n t e s t i c u l a r and caput spermatozoa ( s a g i t t a l s e c t i o n s ) 118 5 5 - 5 8 The movement of the c y t o p l a s m i c d r o p l e t , the adhesiveness of the c e l l membrane to the t i p of the acrosome, and the o c c u r r e n c e of the s a t e l l i t e f i b r i l s . . 120 AC KNOWLE DGEME NTS I would l i k e f i r s t of a l l to express my g r a t i t u d e to Dr. W.D. K i t t s , my s u p e r v i s o r and the Dean of the F a c u l t y of A g r i c u l t u r a l S c i e n c e s , f o r a s s i s t a n c e and encouragement. S i n c e r e thanks are due to Dr. M.S. Ahmad f o r h i s i n v a l u a b l e s u g g e s t i o n s and help d u r i n g the d e s i g n and progress of the study. I take t h i s o p p o r t u n i t y to r e c o r d my thanks to p r o f e s s o r s Dr. C R . K r i s h n a m u r t i , Dr. R.G. Peterson and Dr. M.A. Tung f o r t h e i r a d v i c e and guidance, to Mr. G. Galzy, Mrs. M. S t r i k e r , Mrs. P.K. G i l l and Mr. J . Cole f o r t h e i r t e c h n i c a l a s s i s t a n c e , and to Mrs. G. Huchelega and Miss F.E. Newsome f o r t h e i r generous h e l p on numerous o c c a s i o n s . I would a l s o l i k e to thank Dr. K.H. -Yeon, the P r e s i d e n t of Chung-Buk U n i v e r s i t y i n Korea, P r o f e s s o r H.J. Lee and Dr. H.K. Song f o r t h e i r c o n t i n u e d e f f o r t s i n h e l p i n g and encouraging my progress through the past s e v e r a l y e a r s . T h i s r e s e a r c h was supported by A g r i c u l t u r e Canada (EMR RB-1974-2) and by the N a t i o n a l Research C o u n c i l of Canada (A-132). The A s i a Foundation p r o v i d e d a t r a v e l grant from Seoul to Vancouver f o r the auth o