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

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DEGENERATION OF THE GERMINAL EPITHELIUM IN THE MOUSE AND RAT TESTIS WITH RESPECT TO THE SEMINIFEROUS CYCLE FOLLOWING LIGATION OF THE VASA EFFERENTIA by GEORGE ALBERT LANGFORD B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming t o the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1967 In presenting th is thesis in pa r t ia l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i sh Columbia, I agree that the Library sha l l make i t f ree ly ava i lab le for reference and Study. I further agree that permission for extensive copying of this thesis for scho lar ly purposes may be granted by the Head of my Department or by h.i.<s representat ives. It is understood that copying or publ icat ion of th is thesis for f inanc ia l gain shal l not be allowed without my wr i t ten permission. Department of ZOOLOGY  The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date NOVEMBER 6, 1967 ABSTRACT The manner i n which the c y c l e of the germinal e p i t h e l i u m i s a f f e c t e d d u r i n g p r o g r e s s i v e atrophy of sem-i n i f e r o u s t u b u l e s , and the form of c e l l u l a r degeneration t h a t occurs at each stage of the c y c l e has been studied i n ad u l t r a t and mouse t e s t i s f o l l o w i n g u n i l a t e r a l l i g a t i o n of the vasa e f f e r e n t i a . F o l l o w i n g the l i g a t i o n , which was placed as c l o s e t o the t e s t i s as p o s s i b l e , groups of animals were k i l l e d a t po s t o p e r a t i v e periods ranging from 1 t o 10 days f o r r a t s , and 16 hours t o 7 days f o r mice. The stages of the c y c l e were i d e n t i f i e d by the p e r i o d i c a c i d - S c h i f f - h a e m a t o x y l i n technique using the c l a s s i f i c a t i o n of Leblond and Clermont (1952) and Oakberg (1956a). The t i m i n g of the c y c l e d i d not appear t o be d i s -rupted by the degenerative changes t h a t r e s u l t e d : at a l l p o s t o p e r a t i v e p e r i o d s , the v i a b l e germ c e l l s were a s s o c i -ated w i t h the same stages of the c y c l e as they are i n the normal t e s t i s . The frequency w i t h which each stage of the c y c l e occurred was measured i n the l i g a t e d t e s t i s of the mouse and found t o be unchanged from t h a t e s t a b l i s h e d i n the c o n t r o l t e s t i s . i i The a t r o p h i c r e a c t i o n s at each stage of the c y c l e followed a c o n s i s t e n t and w e l l - d e f i n e d sequence of events which have been described i n the form of a general scheme. The proposed scheme has not p r e v i o u s l y been described i n the l i t e r a t u r e , and c o n s i s t s of 4 main steps i n tubule degener-a t i o n . During stages 1 - IX of the c y c l e these steps are: (1) the "spermatid sloughing" stage, (2) the "multinuc-l e a t e d body" stage, (3) the " S e r t o l i c e l l cytoplasm" stage, and (4) the " S e r t o l i c e l l sloughing" stage. During stages X - X l l of the c y c l e i n the mouse (stages X - XIV i n the r a t ) , the f i r s t 2 of the 4 degenerative steps d i f f e r and have been termed: (1) the " c e l l u l a r sloughing" stage, and (2) the "spermatocyte ghost c e l l " stage. Degeneration of seminiferous tubules a f f e c t e d S e r t o l i c e l l s i n a d e f i n i t e manner: the S e r t o l i c e l l n u c l e i increased i n number, f r e q u e n t l y migrated away from the base-ment membrane and were o f t e n sloughed i n t o the tubule lumen. These changes i n the behaviour of S e r t o l i c e l l s d u r i n g tub-u l a r atrophy are d i s c u s s e d . i i i TABLE OF CONTENTS Abs t r a c t i i Table of Contents i v L i s t of Tables v i Acknowledgements v i i INTRODUCTION 1 MATERIALS AND METHODS 6 1. L i g a t i o n of Vasa E f f e r e n t i a i n Rats 7 11. L i g a t i o n of Vasa E f f e r e n t i a i n Mice 8 111. E l e c t r o n Microscopy 9 RESULTS 13 1. L i g a t i o n of the Vasa E f f e r e n t i a i n Rats 13 11. L i g a t i o n of the Vasa E f f e r e n t i a i n Mice 13 A. Changes i n the Cycle of the Seminif-erous E p i t h e l i u m 13 B. Changes i n the S i z e of Seminiferous Tubules 15 C. Changes i n the Number of S e r t o l i C e l l N u c l e i 17 D. H i s t o l o g i c a l Changes i n the Epididymis 18 i v TABLE OF CONTENTS (Con't) E. H i s t o l o g i c a l Changes i n the Germinal 18 Ep i t h e l i u m 1. Degenerative Steps i n Stages 22 X - X l l of the Cycle 2. Degenerative Steps i n Stages 23 1 - IX of the Cycle 3. The Fate of M u l t i n u c l e a t e d Bodies 26 4. The Fate of R e s i d u a l Bodies 28 F. At r o p h i c Reactions i n Normal Tissue 29 DISCUSSION 30 SUMMARY 38 LITERATURE CITED 41 KEY TO ABBREVIATIONS 46 PLATES V LIST OF TABLES Table 1. The c y c l e of the seminiferous e p i t h e l i u m i n the r a t . 11 Table 2. The c y c l e of the seminiferous e p i t h e l i u m i n the mouse. 12 Table 3. Percentage w i t h which each stage of the seminiferous c y c l e occurs f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a i n the mouse. 14 Table 4. Mean diameter of 20 seminiferous tubules from each p o s t o p e r a t i v e stage i n mice. 16 Table 5. Mean S e r t o l i c e l l counts from 20 semin- 17 i f e r o u s tubule cross s e c t i o n s at each p o s t o p e r a t i v e stage i n mice. Table 6. The h i s t o l o g i c a l changes i n the germinal 20 e p i t h e l i u m f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a i n mice. v i ACKNOWLEDGEMENTS I would l i k e t o express my s i n c e r e thanks t o Dr. A.B. Acton f o r h i s continued support and encouragement, and f o r a l l o w i n g me t o chose the current research problem. Thanks are a l s o due t o Dr. C.V. Finnegan & Dr. P. Ford f o r t h e i r c r i t i c i s m of the manuscript. F i n a l l y , I would l i k e t o acknowledge the i n t e r e s t of Dr. J.A. Birkbeck at the Vancouver General H o s p i t a l . v i i INTRODUCTION Since the description of the cycle of the seminiferous epithelium i n the testes of c e r t a i n mammals i n terms of develop-mental r e l a t i o n s h i p s of a l l germ c e l l s (Leblond and Clermont, 1952), i t i s now possible to study systematically the c e l l u l a r events comprising spermatogenesis - from the e a r l i e s t germ, c e l l s , type A spermatogonia, to morphologically mature sper-matozoa. Previously, no adequate r e l i a b l e method had been devised for c l a s s i f y i n g the d i f f e r e n t c e l l u l a r associations of germ c e l l s (Roosen - Runge and Giesel, 1950). The i n i t i a l report by Leblond and Clermont (1952), stimulated a large number of d e t a i l e d investigations of the cycle of the semin-iferous epithelium i n various animals (Clermont, 1954, 1963; Clermont and Leblond, 1955, 1959; Daoust and Clermont, 1955; Clermont and Perey, 1957; Perey, Clermont and Leblond, 1961; Oakberg, 1955 a,b, 1956 a,b; Sapsford, 1962, a,b), but these reports a l l focus attention on the cycle as applied to sper-matogenesis i n the normal t e s t i s * The exact manner, however, i n which the cycle i s affected during progressive t e s t i c u l a r atrophy, and the type of c e l l u l a r degeneration that occurs at various stages of the cycle, has not been well defined. The purpose of t h i s thesis i s to c l a r i f y these two problems i n the mouse and i n the r a t . 2 The c l a s s i f i c a t i o n of the cycle of the seminiferous epithelium i s based on well defined steps i n the development of spermatids to morphologically mature spermatozoa. D i f f e r -ences i n the acrosomic and head cap regions of spermatids, as seen after periodic a c i d - S c h i f f staining, have made i t possible to describe 19 steps i n the r a t (Leblond and Clermont, 1952), and 16 steps i n the mouse (Oakberg, 1956a). The f i r s t 14 steps of spermatid development i n the r a t , and the f i r s t 12 steps i n the mouse, define the stages of the seminiferous cycle i n each animal. A b r i e f explanation of the cycle with respect to the spermatids i s presented with plate 1. According to Leblond and Clermont, the process of spermatogenesis extends over 4 complete cycles of the seminif-erous epithelium, beginning i n stage IX with mitoses i n type A spermatogonia, and ending with the release of spermatozoa i n stage V l l l of the c y c l e . It i s therefore important to deter-mine how the cycle i s affected during progressive t e s t i c u l a r atrophy, since the factors that are responsible for the devel-opmental re l a t i o n s h i p s of a l l germ c e l l s i n the cycle are also the factors which determine the rate of spermatogenesis. In hypophysectomized r a t s , Clermont and Morgentaler (1955) observed that the cycle of the seminiferous epithelium was not affected, although the numbers of spermatogonia, 3 spermatocytes and spermatids were greatly reduced. This was v e r i f i e d by the radioautographic r e s u l t s of Clermont and Harvey (1965), which showed that the r e l a t i v e durations of each stage of the cycle i n testes from hypophysectomized rat s were e s s e n t i a l l y unchanged from the normal control testes. Recently, Chowdhury and Steinberger (1964) reported that the frequency of occurrence of each stage i n the spermatogenic cycle remains r e l a t i v e l y unchanged when r a t testes are exposed o t o 43 C. At t h i s temperature, however, only a p a r t i a l degen-eration of the germinal epithelium occurs. Whether the cycle i s affected during more severe conditions of atrophy has not been determined. In order to study the exact manner i n which the cycle of the seminiferous epithelium i s affected during a progressive degeneration of the germinal epithelium, a method of inducing t e s t i c u l a r atrophy was adopted which i s not depen-dent on the heat s e n s i t i v i t y of the d i f f e r e n t germ c e l l s , nor on the influence of s p e c i f i c p i t u i t a r y hormones. The method selected was l i g a t i o n of the vasa e f f e r e n t i a . The e f f e c t of t h i s operation on the t e s t i s i n mice has not previously been reported, but e a r l i e r studies of l i g a t i o n of the vasa efferen-t i a i n r a t s (Van Wagenen, 1924, 1925, 1926; Smith, 1962), indicated that a rapid and complete degeneration of the germinal 4 epithelium i s produced. These reports, however, do not con-sider how the spermatogenic cycle i s affected, nor do they describe the type of c e l l u l a r degeneration that occurs at various stages of the cycle i n d e t a i l . An example of a seminiferous tubule undergoing severe atrophy following l i g a t i o n of the vasa e f f e r e n t i a i s shown i n figure 1. F i g . 1. Seminiferous tubule of a r a t f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a f o r 6 days. Extreme atrophy during the e a r l y stages of the c y c l e of the seminiferous e p i t h e l i u m has r e s u l t e d . Numerous m u l t i n u c l e a t e d bodies (MNB) of immature spermatids are found i n the germinal e p i t h e l i u m i n v a r i o u s s t a t e s of degeneration; one m u l t i n u c l e a t e d body at the lumen of the tubule (L) i s about t o be e x f o l i a t e d . Many S e r t o l i c e l l n u c l e i (SC) are d i s p l a c e d away from the basement mem-brane (BM) and occupy a p o s i t i o n adjacent t o m u l t i n u c l e a t e d bodies, n e c r o t i c pachytene spermatocytes (PC), and at the edges of vacuoles (V). Spermatogonia (SG) s t i l l r e t a i n t h e i r normal nuclear morphology and p o s i t i o n adjacent t o the basement membrane. Iron haematoxylin. X 1350. 5 6 MATERIALS AND METHODS The organization of the cycle of the seminiferous epithelium i n the r a t (Leblond and Clermont, 1952), and i n the mouse (Oakberg, 1956 a) has been reproduced i n tables 1, and 2, pages 11 and 12, i n order to provide a reference for the extent of development of the d i f f e r e n t germ c e l l s con-s t i t u t i n g each stage of the c y c l e . The term " c e l l u l a r association" i s synonymous with stage - with respect to the cycle, and includes the type of spermatogonia, spermatocytes and spermatids which make up a p a r t i c u l a r region of a semin-iferous tubule. The v e r t i c a l columns, indicated by Roman numerals i n each table, represent the stages of the cy c l e . The type of spermatogonia and the developmental steps of spermatocytes i n each stage are represented by c a p i t a l l e t t e r s ; the developmental steps of spermatids, i n Arabic numerals; the mitotic d i v i s i o n s of spermatogonia, by sub-s c r i p t m; and the maturation d i v i s i o n s of primary and secondary spermatocytes, by subscript md. The germ c e l l s seen i n a cross section of any given region of a tubule i n the r a t or mouse t e s t i s would therefore represent only one of the c e l l u l a r associations of either table 1 or 2 re s p e c t i v e l y . 7 The animals used i n the present experiments were he a l t h y s e x u a l l y mature male Wistar r a t s (210 - 300 g ) , and male Swiss mice (28 - 35 g ) , maintained i n a c o n t r o l l e d environment room at 23°C. B u c k e r f i e l d 1 s Crumbles and water were s u p p l i e d ad l i b i t u m . The h i s t o l o g i c a l procedure i n v o l v e d f i x a t i o n of t e s t e s and t h e i r a d j o i n i n g epididymis i n Bouin's f l u i d f o r 2 days, fo l l o w e d by four 2-hour r i n s e s i n 70% e t h a n o l . The t i s s u e was dehydrated, c l e a r e d i n benzene, and embedded i n p a r a f f i n a t 59°C, S e r i a l s e c t i o n s 5 microns t h i c k of a l l t e s t e s and i n t a c t epididymides were made, and mounted on g l a s s s l i d e s . Sections were then processed u s i n g the p e r i o d i c a c i d - S c h i f f - haematoxylin method of Leblond and Clermont (1952) f o r i d e n t i f i c a t i o n of the v a r i o u s stages of the c y c l e . An a l t e r n a t e s t a i n i n g method used f o r i d e n t i f i c a t i o n of the stages of the c y c l e was Heidenhain's i r o n haematoxylin f o l l o w e d by b l u e i n g i n l i t h i u m carbonate (Clermont and Perey, 1957) . 1, L i g a t i o n of Vasa E f f e r e n t i a i n Rats The r i g h t t e s t i s of 15 r a t s was d i s p l a c e d from the scrotum through the i n g u i n a l c a n a l i n t o the abdominal c a v i t y 8 and g e n t l y l i f t e d outside the animal through a small supra-p u b l i c i n c i s i o n . The vasa e f f e r e n t i a were l o c a t e d at t h e i r o r i g i n - an avascular o v a l on the surface of the t e s t i s near the caput e p i d i d y m i d i s . The l i g a t i o n was made w i t h a f i n e suture (Measuroll s i l k 000) as c l o s e t o the o r i g i n ; as p o s s i b l e without d i s r u p t i n g the t u n i c a albuginea. The t e s t i s was kept, moist w i t h i s o t o n i c mammalian s a l i n e , and at the completion of the operation i t was replaced along the i n -g u i n a l c a n a l t o the scrotum. In t h i s way, the scrotum r e -mains undisturbed and maintains e s s e n t i a l l y a normal e n v i r o n -ment once the t e s t i s i s r e p l a c e d . The l e f t t e s t i s was used as a c o n t r o l . The r a t s were d i v i d e d i n t o groups of 3 and k i l l e d by means of ether vapour at 1, 2, 4, 6 and 10 day pos t o p e r a t i v e p e r i o d s . Subcutaneous i n j e c t i o n s of c o l c h i c i n e (0.2 mg/100 g body weight, d i s s o l v e d i n 0.5 ml normal s a l i n e ) were given t o each group of animals 6 hours before they were s a c r i f i c e d . 11. L i g a t i o n of Vasa E f f e r e n t i a i n Mice The method used above t o l i g a t e the vasa e f f e r e n t i a i n r a t s , was used t o l i g a t e the vasa e f f e r e n t i a of the r i g h t t e s t i s of 3 5 mice. The mice were then d i v i d e d i n t o groups of 9 5 and s a c r i f i c e d at 9 hour, 16 hour, 24 hour, 2 day, 4 day, 5 day and 7 day po s t o p e r a t i v e p e r i o d s . Counts of the 12 d i f f e r e n t stages of the c y c l e , expressed as percentages of the t o t a l number of stages counted, were made by i d e n t i f y i n g the stage of every seminiferous tubule appearing i n 4 d i f f e r -ent s e c t i o n s of t e s t i s from each of 2, 4 and 7 day postoper-a t i v e p e r i o d s . Measurements of the mean diameter of 20 cross s e c t i o n s of seminiferous tubules were made at each postoper-a t i v e p e r i o d , and from each of these tubules measured, the mean number of S e r t o l i c e l l n u c l e i per cross s e c t i o n of seminiferous tubule was determined. 111. E l e c t r o n Microscopy Small segments of seminiferous tubules about 1 -2 mm i n length from c o n t r o l r a t t e s t e s and r a t t e s t e s l i g a t e d f o r 4 and 6 day periods were f i x e d f o r 1 hour i n 6.5% g l u t a r -aldehyde, phosphate b u f f e r e d t o pH 7.2 at 4°C. A f t e r 3 r i n s e s i n b u f f e r , the t i s s u e was p o s t f i x e d i n phosphate b u f f e r e d 1% osmium t e t r o x i d e , r i n s e d twice i n b u f f e r , dehydrated i n a graded s e r i e s of a l c o h o l s , and embedded i n Epon 812 f o r 12 o o hours at 37 C and then 24 hours at 60 C. Thin s e c t i o n s of both normal and a t r o p h i c tubules were s t a i n e d w i t h u r a n y l 1 0 acetate f o l l o w e d by lead c i t r a t e and examined w i t h a H i t a c h i HS 7S e l e c t r o n microscope. Observations were c o r r e l a t e d w i t h the r e s u l t s of l i g h t microscopy t o a s s i s t i n the i n t e r -p r e t a t i o n of p r o g r e s s i v e atrophy at each stage of the semin-i f e r o u s c y c l e . TABLE 1* The Cycle of the Seminiferous E p i t h e l i u m i n the Rat 15 16 16 17 17 18 19 19 1 2 3 4 5 6 7 8 9 10 11 12 13 14 P P P P P P P P P P P P Dip Dia/S (md) In 1 In In In/B (m) B B/R (m) R R L L L L/Z Z Z A A A A A A A A A (m) A A A (m) A A (m) 1 11 111 IV V VI VI1 V l l l IX X XI X l l X l l l XIV * A f t e r Leblond and Clermont, 1952 m = m i t o t i c d i v i s i o n md = m e i o t i c d i v i s i o n A = type "A" spermatogonia In = type "In" spermatogonia B = type "B" spermatogonia S = secondary spermatocyte R = r e s t i n g spermatocyte L = leptotene spermatocyte Z = zygotene spermatocyte P = pachytene spermatocyte Dip = d i p l o t e n e spermatocyte Dia = d i a k i n e s i s spermatocyte TABLE 2* The Cycle of the Seminiferous E p i t h e l i u m i n the Mouse 11 111 IV VI V l l V l l l IX X XI X l l A A A A A A A A A(m) A (m) A A (m) A/In (m) In In/B (m) B B/R (m) R R/L L L/Z Z Z/P P P P P P P P P P P Dip Dia/S (md) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 14 15 15 15 16 16 * A f t e r Oakberg, 1956 m == m i t o t i c d i v i s i o n md = m e i o t i c d i v i s i o n A = type "A" spermatogonia In = type "In" spermatogonia B = type "B" spermatogonia S = secondary spermatocyte R = r e s t i n g spermatocyte L = leptotene spermatocyte Z = zygotene spermatocyte P = pachytene spermatocyte Dip = d i p l o t e n e spermatocyte D i a = d i a k i n e s i s spermatocyte 13 RESULTS I . L i g a t i o n of the Vasa E f f e r e n t i a i n Rats The degenerative changes i n the germinal e p i t h e l i u m f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a i n r a t s were essent-i a l l y the same as the changes described i n mice below. Examples of r a t seminiferous tubules at s p e c i f i c steps of atrophy (p l a t e s 7 - 1 3 ) have been used together w i t h a t r o p h i c seminiferous tubules from mice ( p l a t e 10) t o i l l u s t r a t e the general steps i n seminiferous tubule degeneration described below. The stages of the c y c l e were more r e a d i l y i d e n t i f i e d by l i g h t microscopy than by e l e c t r o n microscopy. This i s e x e m p l i f i e d by comparing the head cap of spermatids as seen by each method. Figure 14 i l l u s -t r a t e s the head cap of a spermatid during stage V l l l of spermio-genesis as seen by the e l e c t r o n microscope; f i g u r e 9, and at higher m a g n i f i c a t i o n , f i g u r e s 57 and 58 represent the comparative stage, as seen by l i g h t microscopy. I I . L i g a t i o n of the Vasa E f f e r e n t i a i n Mice A. Changes i n the Cycle of the Seminiferous E p i t h e l i u m The frequency w i t h which each stage of the c y c l e appears i n degenerative t e s t e s i s shown i n t a b l e 3, and was 14 TABLE 3 Percentage w i t h which each stage of the seminiferous c y c l e occurs f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a i n mice Stages C o n t r o l 2 days 4 days 7 days 1 11,6 10.4 13.6 13.3 11 9.1 8.3 9.8 3.9 111 4.4 5.4 7.6 3.6 IV 8.2 6.9 4.6 2.4 V 5.1 4.3 5.8 6.3 VI 8.4 7.5 6.7 7.2 V l l 9.4 10.6 9.9 10.1 V l l l 9.8 11.1 10.6 11.2 IX 7.5 8.4 5.3 8.1 X 6.6 5.9 6.1 5.2 XI 9.4 8.2 4.6 7.9 X l l 10.5 11.3 12.0 16.2 Undetermined 0.0 1.7 3.4 4.6 Tubules counted 734 628 486 566 15 used as an i n d i c a t i o n of the e f f e c t s of atrophy on the c y c l e i t s e l f . As atrophy of the germinal e p i t h e l i u m progressed from 2 days through 7 days, i d e n t i f i c a t i o n of each stage of the c y c l e became i n c r e a s i n g l y d i f f i c u l t . Spermatid degener-a t i o n , and i n some tubules the complete lack of spermatids, was extensive enough t o make i t necessary t o i n c l u d e a per-centage of u n c l a s s i f i e d t u b u l e s . With few exceptions, the r e l a t i v e frequency of appearance of each stage of the c y c l e i n a t r o p h i c t e s t e s at a l l p o s t o p e r a t i v e periods analyzed was not s i g n i f i c a n t l y d i f f e r e n t from c o n t r o l f r e q u e n c i e s . No p a r t i c u l a r c e l l u l a r a s s o c i a t i o n was a f f e c t e d more s e r i o u s l y than any other stage, of the c y c l e by appearing a greater or fewer number of times than i n the normal t e s t e s . Severe atrophy of seminiferous tubules d i d not appear t o d i s r u p t the developmental r e l a t i o n s h i p s of the remaining germ c e l l s i n the germinal e p i t h e l i u m . The only d e v i a t i o n s from the c o n t r o l frequencies were: (1) decreases i n stages IV and XI of the 4 day p o s t l i g a t i o n p e r i o d , and stages 11 and IV of the 7 day p e r i o d , and (2) increases i n stage 111 at 4 days, and stage X l l at 7 days. B. Changes i n the Size of Seminiferous Tubules In cross s e c t i o n s of seminiferous tubules there was an i n c r e a s e i n the average diameter of tubules as e a r l y as 16 s i x t e e n hours f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a (table 4 ) . This i n c r e a s e was g r e a t e s t a f t e r the vasa e f f e r e n t i a had been l i g a t e d f o r seven days. Only the m a j o r i t y of tubules demonstrating a s i m i l a r degree of atrophy were considered. TABLE 4 Mean Diameter of 20 Seminiferous Tubules From Each Po s t -o p e r a t i v e Stage F o l l o w i n g Vasa E f f e r e n t i a L i g a t i o n i n Mice Duration of Experimental C o n t r o l l i g a t i o n diameter (u) diameter (u) 16 hour 205 , 180 1 day 195 172 2 day 210 184 4 day 205 176 5 day 215 179 7 day 220 187 The i n c r e a s e i n s i z e of seminiferous tubules was con-cu r r e n t w i t h a s w e l l i n g of the l i g a t e d t e s t i s when compared w i t h the c o n t r a l a t e r a l c o n t r o l . The t u r g i d i t y was maximal between 2 and 5 days p o s t l i g a t i o n . By 7 days p o s t l i g a t i o n , the s i z e of the l i g a t e d t e s t i s had decreased t o about the s i z e of the c o n t r o l t e s t i s . 17 C. Changes i n the Number of S e r t o l i C e l l N u c l e i Counts of S e r t o l i c e l l n u c l e i f o r each of the cross s e c t i o n s of seminiferous tubules used f o r tubule measurements, showed a d e f i n i t e i n crease i n the number of n u c l e i at 2 days p o s t l i g a t i o n ( t a b l e 5 ) . This numerical increase i n n u c l e i p e r s i s t e d u n t i l 5 days p o s t l i g a t i o n , when a marked decrease i n S e r t o l i c e l l n u c l e i r e s u l t e d . Counts of S e r t o l i c e l l n u c l e i i n c o n t r a l a t e r a l t e s t e s showed no apparent change. This numerical increase i n S e r t o l i c e l l n u c l e i was a l s o observed i n r a t s f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a ( p l a t e 9 ) . When these animals were i n j e c t e d subcutaneously w i t h c o l c h i c i n e , however, no i n d i c a t i o n of S e r t o l i c e l l s undergoing m i t o s i s could be detected. Mean S e r t o l i C e l l Counts From 20 Seminiferous Tubule Cross Sections F o l l o w i n g Vasa E f f e r e n t i a L i g a t i o n i n Mice Duration of l i g a t i o n Experimental count C o n t r o l count TABLE 5 1 day 8.7 8.5 2 day 13.0 8.9 4 day 16.8 7.8 5 day 13.4 8.4 7 day 7.6 9.3 18 D. H i s t o l o g i c a l Changes i n the Epididymis The f i r s t change observed i n cross s e c t i o n s of the epididymis f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a was a decrease i n the number of sperm from t h a t observed i n the c o n t r a l a t e r a l c o n t r o l epididymis at 2 days p o s t l i g a t i o n ( f i g . 115). At 4 days p o s t l i g a t i o n ( f i g . 116), some cross s e c t i o n s of the duct midway between the caput e p i d i d y m i d i s and cauda e p i d i d y m i d i s were almost devoid of spermatozoa, while others contained d i s o r g a n i z e d areas of n e c r o t i c spermatozoa and PAS -p o s i t i v e amorphous masses b e l i e v e d t o be mucus. The contents of a few ducts appeared normal. The contents of a l l duct sec-t i o n s appeared abnormal a f t e r 5 days p o s t l i g a t i o n ( f i g . 117). Many regions of the duct were devoid of spermatozoa. At 7 days p o s t l i g a t i o n ( f i g . 118), most regions of the duct midway along the epididymis were devoid of spermatozoa or contained small clumps of n e c r o t i c spermatozoa and mucus. E. H i s t o l o g i c a l Changes i n the Germinal E p i t h e l i u m The degenerative changes i n the germinal e p i t h e l i u m were extremely v a r i e d i n d i f f e r e n t tubules ( f i g . 102). I t was 19 not uncommon t o f i n d a m o r p h o l o g i c a l l y normal, or s l i g h t l y a t r o p h i c tubule adjacent t o a n e c r o t i c tubule as l a t e as 4 days a f t e r the l i g a t i o n was made ( f i g . 103). The degree of atrophy i n each t u b u l e , however, g e n e r a l l y remained the same throughout i t s l e n g t h . This c o n d i t i o n conveniently lends i t s e l f t o a c l o s e a n a l y s i s of the process of atrophy at the d i f f e r e n t stages of the seminiferous c y c l e . The de-generative changes i n the germinal e p i t h e l i u m are summed up i n t a b l e 6, and are i l l u s t r a t e d on p l a t e s 3 - 6 ( f i g . 15 -86). From the a n a l y s i s of the degenerative changes at each stage of the seminiferous c y c l e , i t was p o s s i b l e t o desc r i b e a general scheme f o r the degeneration of a semin-i f e r o u s t u b u l e . The scheme i s based on two d i f f e r e n t h i s t o l o g i c a l p i c t u r e s found i n a t r o p h i c t u b u l e s : the f i r s t , i n stages 1 - IX of the c y c l e ( f i g . 2 - 10); the second, i n stages X - X l l of the c y c l e ( f i g . 11 - 13). A diagram of t h i s scheme i s shown i n f i g u r e 1 - A. The scheme proposed, has not p r e v i o u s l y been described i n the l i t -e r a t u r e . 20 P o s t -l i g a t i o n p e r i o d Spermato-g o n i a ( a l l s tages ) R e s t i n g ( V I - V l l l ) Spermatocyte s L e p t o t e n e & Zygotene ( V l l l - X l l ) TABLE 6 The H i s t o l o g i c a l Changes i n the G e r m i n a l E p i t h e l i u m F o l l o w i n g L i g a t i o n o f the Vasa E f f e r e n t i a i n M i c e Pachytene ( a l l s tages ) Secondary spermatocytes ( s tage X l l ) Spermat ids i n s t e p s 1 - 9 o f s p e r m i o g e n e s i s ( s tages 1 - IX) . v - r a . • ". V 'it ' I ' V " ' -normal' Spermat ids i n s t eps 10 - 16 o f s p e r m i o g e n e s i s ( s tages X — X l l & 1 - V l l l ) S e r t o l i c e l l s ( a l l s tages ) 16 h o u r normal normal n o r m a l n o r m a l -normal i n most t u b u l e s - o c c a s i o n a l s l o u g h i n g - s l o u g h i n g a t s tages IV - V l l l n o r m a l 1 day n o r m a l normal n o r m a l - n o r m a l i n most-, t u b u l e s - r e d u c e d nuBnberl in s tages 1V-V1 (fig»"3&J40,46) - a few " g h o s i ^ / ' j c e l l s normal ( f i g . 82) -normal i n most t u b u l e s - i n c r e a s e d s l o u g h i n g ( f i g . 58) ( f i g . 82):. -most have been r e l e a s e d p r e m a t u r e l y i n t o the lumen ( f i g . 16 ,70 ,76 ) n o r m a l 2 day n o r m a l normal n o r m a l - r e d u c t i o n -:flii .[ number ( f i g . 2 3 ^ ^ , 4 1 , 5 9 ) - s l o u g h i n g du&li ig s tages 1 , 1 V , V 1 1 , ^ : X 1 1 - i n c r e a s e i i i ' : * ' ghost" c e l l s ( f lg^2^i53) r e d u c t i o n i n number ( f i g . 83) - r e d u c t i o n i n number - o c c u r r e n c e o f c h r o m a t i n - r i n g e d n u c l e i ( f i g . 35 ,53) - i n c r e a s e d s l o u g h i n g ( f i g . 35 ,65 - f o r m a t i o n o f MNB ( f i g . 17,59) m -111' - s l o u g h i n g i n most t u b u l e s ( f i g . 17, 2 3 , 4 7 , 7 1 , 7 7 , 8 3 ) - i n c r e a s e i n number o f n u c l e i i n some t u b u l e s 4 , d a y s no changes d e t e c t e d no changes d e t e c t e d -many s t i l l n o r m a l - p y k n o t i c c e l l s common - o c c a s i o n a l s l o u g h i n g - r e d u c t i o n in number ( f i g , 18',$'$$) - i n c r e a s e ih .nuipber o f "ghost" - o f t e n i n ; c o n g e s t e d l u m i n a ( f i g . i 72) - g r e a t e r r e d u c t i o n i n number - s l o u g h i n g -numerous "ghost" c e l l s ( f i g . 84) - g r e a t e r r e d u c t i o n i n number - i n c r e a s e i n s l o u g h i n g ( f i g . 3 6 , 4 8 , 5 4 , 6 0 ) - i n c r e a s e d number o f c h r o m a t i n -r i n g e d n u c l e i ( f i g . 4 2 , 5 4 , 6 6 ) ' • o f f : - v e r y few r e m a i n ( f i g . 2 4 , 3 0 , 3 6 , 4 2 , 48 , 54, 78) - c o m p l e t e l y b l o c k the l u mi n a o f some t u b u l e s ( f i g . 72) - n u m e r i c a l i n c r e a s e i n n u c l e i ( f i g . 7 8 - n u c l e a r m i g r a t i o n s toward lumen ( f i g . 24 ,72 ,78) -some n u c l e i i n l u m i n a ( f i g . 72) - n u c l e a r shape conforms t o edges o f v a c u o l e s , MNB ( f i g . 72,78) 5 days no changes d e t e c t e d no changes d e t e c t e d • l i t t l e change from 4 day p o s t l i g a t i o n p e r i o d - g r e a t e r reduction in number ( fig • j 31 ,61) - a b s e n t inl/S^e t u b u l e s (fig. 4 9 J J ' ^ ; ; i . - "ghost" ^'JULs* I common - n u c l e a r f c a ^ m e i i t a t i o n (fig. 73%ij*0 - s l o u g h i n g & g £ $ g i j > 73) - g r e a t e r r e d u c t i o n i n number - o n l y a few d i v i s i o n s ( f i g . 85) - g r e a t e r r e d u c t i o n i n number - s l o u g h i n g ( f i g . 3 1 , 3 7 , 5 5 , 6 1 ) - c h r o m a t i n - r i n g e d n u c l e i common ( f i g . 55) - r e d u c t i o n i n number o f MNB ( f i g . 55 ,61) 0 r \: - • IV 7 days no changes d e t e c t e d no changes d e t e c t e d • l i t t l e change from 5 day p o s t l i g a t i o n p e r i o d - a b s e n t in&s&met' t u b u l e s >.*•'<• - ' ( f i g . 20g%.-...-; ; - l i t t l e c h a n g e f r o m 5 day p o s t i ^ ^ i t i L o n p e r i o d • l i t t l e change from 5 day p o s t l i g a t i o n p e r i o d - g r e a t e r r e d u c t i o n i n number - s l o u g h i n g i n most t u b u l e s ( f i g . 2 0 , 2 6 , 3 2 , 5 6 , 6 2 ) - a b s e n t or g r e a t l y r e d u c e d i n number i n most t u b u l e s ( f i g . 2 5 , 3 1 , 5 5 , 6 1 79,85) - s l o u g h i n g i n t u b u l e s r e t a i n i n g these c e l l s -numerous n u c l e i a r e s loughed i n t o l u m i n a - ( f i g . 5 3 , 7 3 ) , o t h e r w i s e , s i m i l a r t o t h a t o b s e r v e d a t 4 days p o s t l i g a t i o n - l i t t l e change from t h a t observed a t 5 days p o s t l i g a t i o n -number o f n u c l e i r e d u c e d t o about t h a t o f c o n t r o l t e s t i s -most n u c l e i l o c a t e d a t basement membrane 21 Degenerative Steps of a Seminiferous Tubule Stages 1 - IX of the Cycle 1. Spermatid Sloughing Stage 1. C e l l u l a r Sloughing Stage Stages X - X l l of the Cycle F i g . 1 - A The two v a r i a t i o n s of degeneration i n seminiferous tubules are caused by the presence of d i f f e r e n t types of germ c e l l s i n the r e s p e c t i v e stages of the c y c l e of the seminiferous e p i t h e l i u m . During the i n i t i a l phase of t e s t i c u l a r atrophy i n stages 1 - IX of the c y c l e , the m a j o r i t y of c e l l s i n the germ-i n a l e p i t h e l i u m are spermatids i n steps 1 t o 9 of spermiogenesis; i n stages X - X l l of the c y c l e , the m a j o r i t y of c e l l s are spermatocytes• 2. M u l t i n u c l e a t e d Body Stage 3. S e r t o l i C e l l 4. S e r t o l i C e l l Cytoplasm > Sloughing Stage Stage 2• Spermatocyte Ghost C e l l Stage 22 1. Degenerative Steps i n Stages X - X l l of the Cycle The i n i t i a l step i n the degeneration of seminiferous tubules i n stages X - X l l of the c y c l e has been termed the " c e l l u l a r sloughing" stage ( f i g . 88). During t h i s p e r i o d of c e l l u l a r e x f o l i a t i o n , most of the spermatids and some sper-matocytes are r e l e a s e d i n t o the lumen, l e a v i n g mainly sper-matocytes i n the germinal e p i t h e l i u m t o undergo f u r t h e r degeneration. Seminiferous tubules i n the next stage i n t u b u l a r degeneration, the "spermatocyte ghost c e l l " stage ( f i g . 89, 90), are c h a r a c t e r i z e d by pachytene and d i p l o t e n e spermato-cytes w i t h pyknotic n u c l e i and a cytoplasmic a f f i n i t y f o r p e r i o d i c a c i d - S c h i f f reagent. Some of these spermatocytes are sloughed i n t o the lumen ( f i g . 89), but most undergo de-generation i n the germinal e p i t h e l i u m . Severely n e c r o t i c spermatocytes have been termed "ghost c e l l s " as they have l i t t l e or no nuclear m a t e r i a l present, and have a reduced cytoplasmic a f f i n i t y f o r PAS ( f i g . 90). Very few small m u l t i -nucleated bodies are formed by spermatocytes. The younger generation of leptotene and zygotene spermatocytes appear m o r p h o l o g i c a l l y r e s i s t a n t at the beginning of the "ghost c e l l " stage, but many undergo pyknosis d u r i n g the l a t e phases of 23 t h i s degenerative stage. Secondary spermatocytes i n stage X l l of the c y c l e are e i t h e r sloughed i n t o the lumen or under-go d i v i s i o n . Many d i v i d i n g spermatocytes are i n v a r i o u s s t a t e s of k a r y o r r h e x i s and have acquired a PAS - p o s i t i v e cytoplasm. Numerous vacuoles are formed\in the germinal e p i t h e l i u m . The number of S e r t o l i c e l l n u c l e i i n crease during t h i s stage, and some "migrate" away from the basement membrane. The next two degenerative stages, the " S e r t o l i c e l l cytoplasm" stage, and the " S e r t o l i c e l l sloughing" stage, are analogous t o the l a s t two degenerative stages t h a t occur i n t u b u l a r atrophy d u r i n g stages 1 - IX of the c y c l e ( f i g . 98,99). 2. Degenerative Steps i n Stages 1 - IX of the Cycle The degenerative changes o c c u r r i n g i n stages 1 - IX of the c y c l e g e n e r a l l y f o l l o w the same sequence of events. The i n i t i a l step i s the "spermatid sloughing" stage, c h a r a c t e r -i z e d by a general loosening of the germinal e p i t h e l i u m , and r e s u l t i n g i n sloughing of most of the older generation of spermatids i n steps 13 t o 16 of spermiogenesis ( f i g . 92). Spermatids i n steps 15 and 16 (stages V - V l l l of the c y c l e ) are g e n e r a l l y the f i r s t t o be sloughed i n t o the lumen because of t h e i r c l o s e p r o x i m i t y t o the lumen. Some of the younger 24 generation of spermatids i n steps 1 t o 9 of spermiogenesis are a l s o sloughed i n t o the lumen ( f i g . 91). During the l a t t e r p a r t of the "spermatid sloughing" stage, some of the n u c l e i of the younger generation of spermatids undergo pyknosis, producing nuclear r i n g formations of chromatin. Some regions of seminiferous tubules c o n t a i n a l l n e c r o t i c spermatids ( f i g . 92), but g e n e r a l l y , the p r o p o r t i o n of normal t o n e c r o t i c sper-matids i s v a r i a b l e . There does not seem t o be any c o r r e l a t i o n between the number of n e c r o t i c spermatids and the stage of the c y c l e of the seminiferous e p i t h e l i u m . Some primary spermato-cytes i n pachytene undergo pyknosis and acquire a cytoplasmic a f f i n i t y f o r PAS. S e r t o l i c e l l n u c l e i i n crease i n number and some are d i s p l a c e d away from the basement membrane. The f u s i o n of nuclear membranes,' e i t h e r at the acro-somic or other regions of adjacent spermatid n u c l e i , i n normal or n e c r o t i c spermatids, i n i t i a t e s the next stage of atrophy, the "multinucleated body" stage ( f i g . 93, 94). I n i t i a l l y , many sm a l l m u l t i n u c l e a t e d bodies (MNB) c o n t a i n i n g only s e v e r a l spermatid n u c l e i are formed, but these smaller MNB tend t o fuse together t o form r e l a t i v e l y fewer but much l a r g e r MNB ( f i g . 95). Pachytene and some leptotene spermatocytes undergo degenerative changes as described i n the "spermatocyte ghost c e l l " stage of tubules i n stages X - X l l of the c y c l e ( f i g . 89). MNB c o n t a i n -i n g spermatocytes are very seldom formed. As these MNB and 25 n e c r o t i c spermatocytes disappear, vacuoles of v a r y i n g s i z e s are l e f t i n the germinal e p i t h e l i u m ( f i g . 97). O c c a s i o n a l l y , the germinal e p i t h e l i u m of some tubules i s g r e a t l y reduced i n c e l l u l a r content, and the lumen extremely conjested w i t h spermatozoa, loose c e l l s and c e l l u l a r d e b r i s ( f i g . 96)• During the MNB stage, S e r t o l i c e l l s appear a c t i v e l y engaged i n phagocytosis: numerous "migrating" S e r t o l i c e l l n u c l e i are l o c a t e d near or at the edges of vacuoles, MNB, and a t r o p h i c spermatocytes. The cytoplasm of the S e r t o l i c e l l s toward the end of t h i s stage becomes r e l a t i v e l y f r e e of c e l l u l a r compon-ents ( f i g . 97). The succeeding " S e r t o l i c e l l cytoplasm" stage of atrophy ( f i g . 98) i s a marked change from the previous MNB stage. The germinal e p i t h e l i u m contains e s s e n t i a l l y only spermatogonia adjacent t o the basement membrane; o c c a s i o n a l r e s t i n g , leptotene or e a r l y pachytene primary spermatocytes -some of which appear m o r p h o l o g i c a l l y normal; very few sper-matids; phagocytized c e l l u l a r d e b r i s ; and an increased number of S e r t o l i ; c e l l n u c l e i , many of which are l o c a t e d away from the basement membrane. Many lumina of seminiferous tub-u l e s at t h i s stage of atrophy are occupied by S e r t o l i c e l l cytoplasm, so t h a t f r e q u e n t l y the lumina are almost n o n e x i s t -ent ( f i g . 97). 26 As a t r o p h i c c o n d i t i o n s become more severe, the germinal e p i t h e l i u m becomes correspondingly t h i n n e r as a r e s u l t of " S e r t o l i c e l l sloughing" - the next stage i n the sequence of a t r o p h i c steps ( f i g . 99). During t h i s p e r i o d , l a r g e masses of vacuolated S e r t o l i c e l l s , phagocytozed i n -c l u s i o n s , and o c c a s i o n a l l y , spermatids and spermatocytes are sloughed i n t o the r e l a t i v e l y empty t u b u l a r lumen. This ex-f o l i a t i o n appears t o be caused by extensive v a c u o l a t i o n i n the S e r t o l i c e l l cytoplasm near the basement membrane ( f i g . 100), which leads e v e n t u a l l y t o a s e p a r a t i o n from the remain-der of the germinal e p i t h e l i u m adjacent t o the basement mem-brane. E x f o l i a t e d S e r t o l i c e l l s i n the lumens of s e m i n i f e r -ous tubules ( f i g . 101) undergo c e l l u l a r degeneration as do other c e l l s which become separated from the germinal e p i t h -elium. As a r e s u l t of the sloughing, the germinal e p i t h e l i u m becomes g r e a t l y reduced i n s i z e and c o n s i s t s only of spermato-gonia, S e r t o l i c e l l s , and the o c c a s i o n a l primary spermatocyte. 3. The Fate of M u l t i n u c l e a t e d Bodies The most s t r i k i n g d e v i a t i o n from the normal t u b u l a r morphology during atrophy i s the d i s p o s i t i o n of spermatids i n stages 1 - IX of the c y c l e i n both r a t s and mice by formation 2 7 of m u l t i n u c l e a t e d bodies (MNB). Most of these MNB undergo degeneration i n the germinal e p i t h e l i u m . Four stages of MNB degeneration can be d i f f e r e n t i a t e d w i t h i r o n haematoxylin. I n i t i a l l y , MNB c o n t a i n m o r p h o l o g i c a l l y normal spermatid n u c l e i ( f i g . 94), n e c r o t i c spermatid n u c l e i , or a combination of both. The i n f r e q u e n t occurrence of these MNB c o n t a i n i n g apparently normal spermatids suggests t h a t normal n u c l e i degenerate r a p i d l y i n MNB t o nuclear r i n g formations. MNB t y p i c a l l y c o n s i s t of spermatid n u c l e i w i t h i n t a c t mem-branes l o c a t e d at the p e r i p h e r y , and a c e n t r a l cytoplasmic mass ( f i g . 104, 105). Stage 1 MNB c o n t a i n the most hyper-chromatic spermatid n u c l e i . A marked r e d u c t i o n i n nuclear and cytoplasmic s t a i n i n t e n s i t y c h a r a c t e r i z e s stage 2 MNB ( f i g . 104, 105). The nuclear membranes of the spermatids are swollen but i n t a c t • Stage 3 MNB ( f i g . 104) are c h a r a c t e r i z e d by a d i s r u p t i o n of the spermatid nuclear membranes causing a r e d u c t i o n of nuclear s t a i n i n g . Stage 3 MNB have been termed "multinucleated ghosts" (MNG). Late stages of MNG become g r e a t l y d i s o r g a n i z e d ( f i g . 105). The f i n a l stage of MNB n e c r o s i s i n v o l v e s complete l y s i s of the remaining c e l l u l a r mass. The disappearance of 28 the MNG from the germinal e p i t h e l i u m i s concurrent w i t h the formation of vacuoles w i t h i n the germinal e p i t h e l i u m ( f i g . 105). O c c a s i o n a l l y , MNB are sloughed i n t o the tubule lumen d u r i n g any one of the 4 stages of degeneration ( f i g . 105). 4. The Fate of Residule Bodies R e s i d u a l cytoplasmic bodies ( f i g . 106), discarded by spermatids at step 16 of spermiogenesis i n the mouse, and step 19 i n the r a t (stages V l l and V l l l of the c y c l e ) , f o l l o w a s i m i l a r degenerative process as MNB d u r i n g atrophy of the germinal e p i t h e l i u m . In order t o observe the degeneration of r e s i d u a l bodies, seminiferous tubules were l o c a t e d i n which spermiogenesis had continued t o step 16 (step 19 i n the r a t ) but at which p o i n t , a t r o p h i c c o n d i t i o n s of the p a r t i c u l a r tubule had i n h i b i t e d f u r t h e r spermatid development. These tubules i n i t i a l l y c o n t a i n MNB comprised of stage V l l or V l l l spermatids, and l a r g e numbers of r e s i d u a l bodies (RB) d i s t r i b u t e d randomly i n the cytoplasm of S e r t o l i c e l l s ( f i g . 107). The RB are then organized i n t o " r e s i d u a l body masses" (RBM) so t h a t only o c c a s i o n a l l y are RB seen alone i n S e r t o l i c e l l s ( f i g . 108, 109), RBM undergo a s i m i l a r l y s i s as m u l t i n u c l e a t e d bodies, l e a v i n g r e s i d u a l vacuoles i n the germinal e p i t h e l i u m . 29 F. A t r o p h i c Reactions i n Normal Tissue In n e a r l y a l l c o n t r o l t e s t e s analyzed, the h i s t o -l o g i c a l appearance of the germinal e p i t h e l i u m appeared normal. An exception occurred i n a c o n t r o l mouse t e s t i s taken at 1 day p o s t l i g a t i o n . . The t e s t i c u l a r h i s t o l o g y appeared normal except i n one p a r t i c u l a r seminiferous tubule at consecutive stages X l l and 1 of the c y c l e . The remaining stages along the length of the tubule appeared normal. Tubular cross s e c t i o n s of the a t r o p h i c r e g i o n c o n t a i n i n g these 2 stages revealed m u l t i -nucleated bodies c o n s i s t i n g of secondary interphase spermato-cytes at stage X l l of the c y c l e ( f i g . I l l , 112), and step 1 spermatids at stage 1 ( f i g . 110). These MNB occupied only a p a r t i a l r e g i o n of the t u b u l e , while the remainder of the tubule appeared normal. Stage 1 spermatids and secondary spermato-cytes i n interphase were a l s o seen i n the lumen. The other germ c e l l s of the germinal e p i t h e l i u m appeared un a f f e c t e d . 30 DISCUSSION The r e s u l t s of the present study on seminiferous tubule degeneration c l e a r l y show the f o l l o w i n g : (1) the t i m i n g of the c y c l e does not appear t o be d i s r u p t e d : at a l l p o s t o p e r a t i v e p e r i o d s , the v i a b l e germ c e l l s are a s s o c i a t e d w i t h the same stages of the c y c l e as they are i n the normal t e s t i s , (2) the frequency w i t h which each stage of the c y c l e occurs i n the mouse (not measured i n the r a t ) does not change, (3) a d e f i n i t e sequence of n e c r o t i c events p e c u l i a r t o each stage of the c y c l e occurs, and (4) the number and p o s i t i o n of the S e r t o l i c e l l n u c l e i change. One of the main p r o p e r t i e s of the normal t e s t i s i s the s t a b i l i t y of the seminiferous c y c l e . The mechanism f o r t h i s exact t i m i n g during the p r o l i f e r a t i o n of germ c e l l s has not yet been demonstrated. Previous s t u d i e s , however, on t e s t i c u l a r atrophy induced by l e s s severe methods (Clermont and Morgentaler, 1955) i n hypophysectomized r a t s , and i n r a t s f o l l o w i n g an increase i n the i n t r a t e s t i c u l a r temperature (Chowdhury and Steinberger, 1964), agree w i t h the present observations i n mice (tabl e 3) t h a t the frequency d i s t r i b u t i o n of the stages of the c y c l e does not change. Under the 31 e x t e n s i v e degenerative c o n d i t i o n s of the present experiments, then, the p e r s i s t e n c e of the same c e l l u l a r a s s o c i a t i o n s , and the same frequency d i s t r i b u t i o n of stages suggests t h a t the r e g u l a t i o n of the c y c l e i s an inherent g e n e t i c c o n t r o l . Such a c o n t r o l would account f o r regions i n a t r o p h i c tubules which c o n t a i n n e c r o t i c spermatids and spermatocytes beside apparently normal c e l l u l a r c ounterparts. L i g a t i o n of the vasa e f f e r e n t i a must a f f e c t the germinal e p i t h e l i u m i n a n o n s p e c i f i c manner si n c e degenerative changes t o spermatids and spermatocytes occurred i n v a r i a b l e numbers at a l l stages of the c y c l e ( t a b l e 6 ) • The cause of t h i s atrophy has been suggested by Smith (1962) t o be the r e -s u l t of increased i n t r a t e s t i c u l a r pressure due t o the accumu-l a t i o n of i n t r a t u b u l a r f l u i d and germinal products. This would account f o r the increase i n the s i z e of the seminiferous tubules s h o r t l y a f t e r the l i g a t i o n was made (t a b l e 4 ) . A t o t a l blockage of the excurrent ducts was v e r i f i e d f o r each t e s t i s by observing a decrease i n the contents of the epididymal duct ( f i g . 113 -118). Previous s t u d i e s on n o n s p e c i f i c degeneration of the germinal e p i t h e l i u m induced by other methods, have reported a t r o p h i c c o n d i t i o n s of the t e s t i s at v a r i o u s time i n t e r v a l s f o l l o w i n g the i n i t i a l i n d u c t i o n of the atrophy ( a r t i f i c i a l 32 c r y p t o r c h i d i s m : Clegg, 1963 a,b; Davis and F i r l i t , 1966; Niemi and Korraano, 1965; exposure of t e s t i s t o h i g h tempera-t u r e s : Steinberger and Dixon, 1959; t e s t i c u l a r ischaemia: O e t t l e and H a r r i s o n , 1952). The d i f f i c u l t y i n i n t e r p r e t i n g these r e s u l t s i s t h a t the r a t e at Which d i f f e r e n t seminiferous tubules undergo degeneration v a r i e s , c r e a t i n g numerous s t a t e s of atrophy. The present d e s c r i p t i o n of the degenerative steps which a seminiferous tubule undergoes at each stage of the seminiferous c y c l e i s t h e r e f o r e an important guide. By u s i n g t h i s i n f o r m a t i o n , the course of previous degenerative changes can be determined i n any n e c r o t i c t u b u l e . The c l a s s i f i c a t i o n of the steps of seminiferous tubule degeneration i n the mouse i s based on the type of a t r o p h i c r e a c t i o n which a f f e c t s the m a j o r i t y of germ c e l l s at each stage of the c y c l e ( f i g . 1 - A ) . A c c o r d i n g l y , the stages of the c y c l e were d i v i d e d i n t o two main groups: Group 1, con-s i s t i n g of stages 1 - IX of the c y c l e ; and Group 2, c o n s i s t i n g of stages X - X l l (X - XIV i n the r a t ) . The d i v i s i o n i n t o the 2 groups of stages i s a r e s u l t of the morphological changes i n the spermatid nucleus d u r i n g l a t e stage IX of the c y c l e . The marked t r a n s f o r m a t i o n of the r e l a t i v e l y round spermatid nucleus t o the elongate curved head of the spermatozoon appears t o 33 a l t e r the response of the more mature spermatids. Instead of forming m u l t i n u c l e a t e d bodies as the younger generation of spermatids do d u r i n g a t r o p h i c c o n d i t i o n s , the o l d e r spermatids are sloughed i n t o the lumen - l e a v i n g the spermatids i n stages 1 - IX, and the spermatocytes i n stages X - X l l t o undergo n e c r o t i c changes. Seminiferous tubules s i m i l a r t o those des-c r i b e d i n the " S e r t o l i c e l l cytoplasm" stage ( f i g . 98), have been reported i n r a t t e s t i s f o l l o w i n g 30 days of a r t i f i c i a l l y induced c r y p t o r c h i d i s m (Clegg, 1963; Davis and F i r l i t , 1966). Tubules of the " S e r t o l i c e l l sloughing" stage ( f i g . 99, 100), however, have not been reported before i n t e s t e s subjected t o e i t h e r l i g a t i o n of the vasa e f f e r e n t i a or a r t i f i c i a l l y induced c r y p t o r c h i d i s m . In tubules observed beyond the "multinucleated body" stage ( f i g . 104) or the "spermatocyte ghost c e l l " stage ( f i g . 89, 90) of tubule degeneration, pachytene primary spermatocytes, i n g r e a t l y reduced numbers, were the most advanced stages of germ c e l l s i n the germinal e p i t h e l i u m . This has a l s o been noted i n c r y p t o r c h i d r a t t e s t e s which have undergone severe degeneration (Davis and F i r l i t , 1966). The process of spermatogenesis would t h e r e f o r e appear t o be i n h i b i t e d from proceeding beyond the pachytene stage of primary spermatocytes i n tubules which are 34 w e l l i n t o or past the above 2 steps of degeneration. In a l l steps of tu b u l a r atrophy, the degeneration of germ c e l l s i s i n t i m a t e l y a s s o c i a t e d w i t h an increased a c t i v i t y of the S e r t o l i c e l l s . A c o n s i d e r a t i o n of the e f f e c t s of degen-e r a t i o n on S e r t o l i c e l l s i s t h e r e f o r e important. The present experiments i n d i c a t e t h a t 3 marked changes occur t o S e r t o l i c e l l s d u r i n g atrophy: a numerical in c r e a s e i n t h e i r n u c l e i ( t a b l e 5 ) , a m i g r a t i o n of t h e i r n u c l e i away from the basement membrane ( f i g . 98), and f i n a l l y , a decrease i n the number of t h e i r n u c l e i . In normal t e s t e s of r a t s and mice, the complement of S e r t o l i c e l l s i s reached s h o r t l y a f t e r b i r t h and m i t o t i c d i v -i s i o n s cease (Sapsford, 1962). A few i n v e s t i g a t i o n s on t e s t i -c u l a r atrophy though, have reported an increase i n the number of S e r t o l i c e l l n u c l e i i n degenerating seminiferous tubules ( i n hypophysectomized r a t s : Clermont and Morgentaler, 1955; i n hypophysectomized r a t s t r e a t e d w i t h f o l l i c l e s t i m u l a t i n g hormone: Murphy, 1965; i n a r t i f i c i a l l y induced c r y p t o r c h i d r a t t e s t e s : Clegg, 1963). Clermont and Morgentaler o f f e r no exp l a n a t i o n f o r t h e i r o bservations, but Clegg a t t r i b u t e s the numerical increase of S e r t o l i c e l l n u c l e i t o a m i t o t i c d i v i s i o n s . Recent experiments w i t h r a t c r y p t o r c h i d t e s t e s by Davis and F i r l i t (1966), however, i n d i c a t e only an increase i n S e r t o l i 35 c e l l s i z e . There have a l s o been previous r e p o r t s , according t o Nebel and Murphy (1960), of an apparent m i t o t i c p r o l i f e r -a t i o n of S e r t o l i c e l l s f o l l o w i n g i r r a d i a t i o n of r a t t e s t e s . Nebel and Murphy a t t r i b u t e t h i s i n c r e a s e i n S e r t o l i c e l l s t o the formation of " r e s t i t u t i o n " c e l l s . These c e l l s are p e c u l i a r t o i r r a d i a t e d t e s t e s , and are produced as a r e s u l t of an a r r e s t at metaphase of. primary spermatocytes. The p o s s i b i l i t y of S e r t o l i c e l l p r o l i f e r a t i o n d u r i n g t e s t i c u l a r atrophy i s t h e r e -f o r e s t i l l i n doubt. The mechanism f o r the numerical increase i n n u c l e i observed i n the present experiments has not yet been determined. The p o s s i b i l i t y of m i t o t i c p r o l i f e r a t i o n of S e r t o l i c e l l s however, cannot be r u l e d out by the apparent absence of m i t o t i c f i g u r e s i n S e r t o l i c e l l n u c l e i i n r a t t e s t e s t r e a t e d w i t h c o l c h i c i n e . Further i n v e s t i g a t i o n s using t r i t i a t e d thymidine are now being c a r r i e d out. The p o s s i b l e stimulus f o r the changes i n the S e r t o l i c e l l behaviour d u r i n g a t r o p h i c c o n d i t i o n s could be the l a r g e increase i n phagocytic m a t e r i a l ingested by S e r t o l i c e l l s . The phagocytic c a p a c i t y of S e r t o l i c e l l s i s w e l l known i n normal t e s t e s (Smith and Lacy, 1959; Niemi and Kormano, 1965) and a t r o p h i c t e s t e s (Lacy, 1960, 1962; Reddy and Svoboda, 1967). In S e r t o l i c e l l s undergoing a c t i v e phagocytosis of degenerating 36 germ c e l l s , Reddy and Svoboda have demonstrated an immediate inc r e a s e i n the number of lysosomes, and a 40 percent increase i n a c i d phosphatase content. The r o l e of S e r t o l i c e l l s d u r i n g t e s t i c u l a r atrophy then, seems t o be d i r e c t e d mainly toward phagocytosis of degenerating germ c e l l s . One observation of S e r t o l i c e l l behaviour i n the present experiments which does i n d i c a t e a change i n t h e i r r e -a c t i o n t o ingested m a t e r i a l , i s t h e i r d i s p o s i t i o n of r e s i d u a l b o d i e s . During normal spermatogenesis, most r e s i d u a l bodies are r e l e a s e d i n t o the lumen. A few are r e t a i n e d i n the germ-i n a l e p i t h e l i u m , phagocytized by S e r t o l i c e l l s and migrate toward the basement membrane (Niemi and Kormano, 1965; Lacy, 1959; D i e t e r t , 1966). Some migrate independently t o the per i p h e r y of the tubule v i a i n t e r c e l l u l a r spaces ( F i r l i t and Davis, 1965). During seminiferous tubule degeneration, r e s i d -u a l bodies are phagocytized i n greater numbers by S e r t o l i c e l l s , but f a i l t o migrate t o the basement membrane. Instead, they are organized i n t o " r e s i d u a l body masses" ( f i g . 107, 108, 109). The increase i n S e r t o l i n u c l e i c o i n c i d e s w i t h the nuc l e a r "migrations" of S e r t o l i c e l l s away from the basement membrane. Whether S e r t o l i c e l l s become separated from the 37 basement membrane during t h e i r nuclear "migrations" has not been determined. A p o s s i b i l i t y - i f the increase i n S e r t o l i n u c l e i i s by mitosis, i s that the newly formed S e r t o l i c e l l s are displaced toward the lumen and do not come i n t o contact with the basement membrane. During severe atrophic conditions then, these "migrating" S e r t o l i c e l l s would be sloughed in t o the lumen ( f i g . 99, 100). S e r t o l i c e l l sloughing would ac-count f o r the numerical decrease i n S e r t o l i c e l l n u c l e i at 7 days p o s t l i g a t i o n (table 5), and could also account f o r Clegg*s observation of a reduction i n S e r t o l i c e l l number following 28 days of a r t i f i c i a l l y induced cryptorchidism. The p a r t i a l degeneration of the germinal epithelium observed i n one tubule of a control t e s t i s ( f i g . 110, 111), cannot be explained, but the form of the degeneration i s of in t e r e s t . According to Perey et a l (1961), each stage of the cycle can be divided i n t o substages i n which the component germ c e l l s develop independently. The degeneration of only part of the cross sections of t h i s tubule could therefore represent the l i m i t s of the substage comprising the p a r t i c -ular stage of the cycle involved. If t h i s i s true, then i t i s possible that substages of stages i n the seminiferous cycle undergo independent degenerative reactions during t e s t i c u l a r atrophy. 38 SUMMARY 1. The a t r o p h i c r e a c t i o n of the germinal e p i t h -elium t h a t f o l l o w s l i g a t i o n of the vasa e f f e r e n t i a i n a d u l t mice and r a t t e s t e s was studi e d at each stage of the seminiferous c y c l e at v a r i o u s p o s t l i g a t i o n p e r i o d s . 2. The t i m i n g of the c y c l e d i d not appear t o be d i s r u p t e d by the degenerative changes t h a t r e s u l t e d : at a l l p o s t o p e r a t i v e p e r i o d s , the v i a b l e germ c e l l s were ass o c i a t e d w i t h the same stages of the c y c l e as they are i n the normal t e s t i s . 3. The frequency w i t h which each stage of the c y c l e occurred was measured i n the l i g a t e d t e s t i s of the mouse and found t o be unchanged from t h a t e s t a b l i s h e d i n the c o n t r o l t e s t i s . 4. The a t r o p h i c r e a c t i o n s at each stage of the c y c l e followed a c o n s i s t e n t and w e l l - d e f i n e d sequence of events. These have been described i n the form of a general scheme f o r tubule degeneration. The proposed scheme c o n s i s t s of 4 main steps which, duri n g stages 1 -IX of the c y c l e , are: (1) the "spermatid sloughing" 39 stage, (2) the "multinucleated body" stage, (3) the " S e r t o l i c e l l cytoplasm" stage, and (4) the " S e r t o l i c e l l sloughing" stage. During stages X - X l l of the c y c l e i n the mouse (stages X - XIV i n the r a t ) , the f i r s t 2 of the 4 degenerative steps d i f f e r and have been termed: (1) the " c e l l u l a r sloughing" stage, and (2) the "spermatocyte ghost c e l l " stage. 5. Spermatogenesis was i n h i b i t e d from proceeding f u r t h e r than pachytene spermatocytes i n tubules which were w e l l i n t o or beyond the "multinucleated body" stage or the "spermatocyte ghost c e l l " stage of tubule degeneration. 6. Four steps i n the degeneration of m u l t i n u c l e a t e d bodies of spermatids have been d i f f e r e n t i a t e d and d e s c r i b e d . 7. Cytoplasmic r e s i d u a l bodies, sloughed by mature spermatids p r i o r t o severe t u b u l a r atrophy, were organized i n t o " r e s i d u a l body masses" w i t h i n the S e r t o l i c e l l cytoplasm. These r e s i d u a l body masses then followed a s i m i l a r l y s i s as m u l t i n u c l e a t e d bodies. 8. Degeneration of seminiferous tubules a f f e c t e d S e r t o l i c e l l s i n a d e f i n i t e manner: the S e r t o l i c e l l 40 n u c l e i increased i n number, f r e q u e n t l y migrated away from the basement membrane and f i n a l l y , decreased i n number as a r e s u l t of S e r t o l i c e l l sloughing i n t o the lumen. 41 LITERATURE CITED Chowdhury, A.K., Steinberger, E., 1964 "A q u a n t i t a t i v e study of the e f f e c t of heat on germinal e p i t h e l i u m of r a t t e s t e s . " Am. J . Anat., 115: 509 - 524. Clegg, E.J., MacMillan, E.W.,. 1965 "The uptake of v i t a l dyes and p a r t i c u l a t e matter by the S e r t o l i c e l l s of the r a t t e s t i s . " J . Anat., Lond., 99.: 219 - 229. Clegg, E.J., 1963 "Observations on the uptake of I n d i a ink by the seminiferous tubules and excurrent duct systems of the r a t t e s t e s . " J . Anat., Lond., 97: 154. Clegg, E.J., 1963 "Studies on a r t i f i c i a l c r y p t o r c h i d i s m : morphological and q u a n t i t a t i v e changes i n the S e r t o l i c e l l s of the r a t t e s t i s . " J . E n d o c r i n o l . , 26: 567 - 574. Clegg, E.J., 1963 "Studies on a r t i f i c i a l c r y p t o r c h i d i s m : degenerative and r e g e n e r a t i v e changes i n the germinal e p i t h e l i u m of the r a t t e s t i s . " J . E n d o c r i n o l . , 27: 241 - 251. Clermont, Y., Harvey, S.C., 1965 "Duration of the c y c l e of the seminiferous e p i t h e l i u m of normal, hypophysectom-i z e d and hypophysectomized - hormone t r e a t e d a l b i n o r a t s . " J . E n d o c r i n o l . , 76: 80 - 89. Clermont, Y., 1963 "The c y c l e of ,the seminiferous e p i t h -elium i n man." Am. J . Anat., 112: 35. Clermont, Y., Leblond, C P . , 1959 " D i f f e r e n t i a t i o n and renewal of spermatogonia i n the monkey, Macacus rheous." Am. J . Anat., 104: 237 - 273. Clermont, Y., Perey B,, 1957 "The stages of the c y c l e of the seminiferous e p i t h e l i u m of the r a t : p r a c t i c a l d e f i n i t i o n s i n PA-Schiff-hematoxylin and hematoxylin-42 e o s i n s t a i n e d s e c t i o n s . " Rev. Canad. B i o l . , 16: 451 - 462. Clermont, Y., Perey, B., 1957 " Q u a n t i t a t i v e study of the c e l l p o p u l a t i o n of the seminiferous tubules i n immature r a t s . " Am. J . Anat., 100: 241 - 267. Clermont, Y., Morgentaler, H»? 1955 " Q u a n t i t a t i v e study of spermatogenesis i n the hypophysectomized r a t . " Endocrinology, 5J7: 369 - 382. Clermont, Y., Leblond, C P . , 1955 "Spermiogenesis of man, monkey, ram and other mammals as shown by the ' p e r i o d i c a c i d - S c h i f f technique." Am. J . Anat., 96.: 229 - 253. Clermont, Y., 1954 "Cycle de l'epithe'lium seminal e t mode de renouvellement des spermatogonies chez l e hamster." Revue Canadienne de B i o l o g i e , 13_: 208 - 245. Clermont, Y., Leblond, C P . , 1953 "Renewal of spermato-gonia i n the r a t . " Am. J . Anat., 93.: 475 - 501. Daoust, R., Clermont, Y., 1955 " D i s t r i b u t i o n of n u c l e i c a c i d s i n germ c e l l s d u r i n g the c y c l e of the s e m i n i f e r -ous e p i t h e l i u m i n the r a t . " Am. J . Anat., 96: 255 - 283. Davis, J.R., F i r / l i t , C.F., 1966 "The germinal e p i t h e l i u m of c r y p t o r c h i d t e s t e s e xperimentally induced i n p r e -p u b e r t a l and a d u l t r a t s . " F e r t i l and S t e r i l . , 17: 187 - 200. D i e t e r t , S.E., 1966 "Fine s t r u c t u r e of the formation and f a t e of the r e s i d u a l bodies of mouse spermatozoa w i t h evidence f o r the p a r t i c i p a t i o n of lysosomes." J . Morph., 120: 317 - 346. F i r l i t , C.F., Davis, J.R., 1965 "Morphogenesis of the r e s i d u a l body of the mouse t e s t i s . " Quart. J . M i c r . S c i . , 106: 93 - 98. Lacy, p., L o f t s , B., 1965 "Studies on the s t r u c t u r e and f u n c t i o n of mammalian t e s t i s . 1. C y t o l o g i c a l and 43 h i s t o c h e m i c a l observations a f t e r continuous treatment w i t h oestrogenic hormone and the e f f e c t s of F.S.H. and L.H." Proc. Roy. Soc. B., 162: 188 - 197. Lacy, D., 1962 " C e r t a i n aspects of t e s t i s s t r u c t u r e and f u n c t i o n . " B r i t . Med. B u l l . , 18: 205 - 208. Lacy, D., 1960 "Light and e l e c t r o n microscopy and i t s use i n the study of f a c t o r s i n f l u e n c i n g spermatogenesis i n the r a t . " J . Roy. M i c r . S o c , 79: 209 - 225. Leblond, C P . , Clermont, Y., 1952 " D e f i n i t i o n of the stages of the c y c l e of the seminiferous e p i t h e l i u m of f t h e r a t . " Ann. N.Y. Acad. S c i . , 55: 548 - 573. Murphy, H.D., 1965 " S e r t o l i c e l l s t i m u l a t i o n f o l l o w i n g i n t r a t e s t i c u l a r i n j e c t i o n s of F.S.H. i n the hypophysec-tomized r a t . " P r o c , Soc. Exp. B i o l . Med., 118(4): 1202 - 1205. Nebel, B.R., Murphy, C.J., 1960 "Damage and recovery of mouse t e s t i s a f t e r lOOOr acute l o c a l i z e d X - i r r a d i a t i o n , w i t h reference t o r e s t i t u t i o n c e l l s , S e r t o l i c e l l i n -crease, and type A spermatogonial recovery." Rad. Res., 12: 626 - 641. Niemi, M., Kormano, M., 1965 " C y c l i c a l changes i n and s i g -n i f i c a n c e of l i p i d s and a c i d phosphatase a c t i v i t y i n the seminiferous tubules of the r a t t e s t i s . " Anat. Rec., 151: 159 - 171. Niemi, M. Kormano, M., 1965 "Response of the c y c l e of the seminiferous e p i t h e l i u m of the r a t t e s t i s t o a r t i f i c i a l c r y p t o r c h i d i s m . " F e r t i l , and S t e r i l . , 16: 236 - 241. Oakberg, E.F., 1956(a) "A d e s c r i p t i o n of spermiogenesis i n the mouse and i t s use i n a n a l y s i s of the c y c l e of the seminiferous e p i t h e l i u m and germ c e l l renewal." Am. J . Anat., 99(3): 391 - 413. Oakberg, E.F. 1956(b) "Duration of spermatogenesis i n the mouse and t i m i n g of stages of the c y c l e ;of the s e m i n i f -erous e p i t h e l i u m . " Am. J . Anat., 99_: 507 - 516. 44 Oakberg, E.F., 1955(a) " S e n s i t i v i t y and time of degener-a t i o n of spermatogenic c e l l s i r r a d i a t e d i n v a r i o u s stages of maturation i n the mouse." Rad. Res., 2.: 369 - 391. Oakberg, E.F., 1955(b) "Degeneration of spermatogonia of the mouse f o l l o w i n g exposure t o X-rays, and stages i n the m i t o t i c c y c l e at which c e l l death occurs." J . Morph., 97.: 39 - 54. O e t t l e , A.G., H a r r i s o n , R.G., 1952 " H i s t o l o g i c a l changes produced i n the r a t t e s t i s by temporary and permanent o c c l u s i o n of the t e s t i c u l a r a r t e r y . " J . Path. Bact., 64: 273 - 297. Perey, B., Clermont, Y., Leblond, C P . , 1961 "The wave of the seminiferous e p i t h e l i u m i n the r a t . " Am. J . of Anat., 108: 47 - 78. Reddy, K.J., Svoboda, D.J., 1967 "Lysosomal a c t i v i t y i n S e r t o l i c e l l s of normal and degenerating seminiferous e p i t h e l i u m of r a t t e s t e s . " Amer. J . Path., 51: 1 - 17. Roosen-Runge, E.C., 1952 " K i n e t i c s of spermatogenesis i n mammals." Ann. N.Y. Acad. S c i . , 55: 574 - 584. Roosen-Runge, E.C, 1951 " Q u a n t i t a t i v e s t u d i e s on sper-matogenesis i n the a l b i n o r a t . 11. The d u r a t i o n of spermatogenesis and some e f f e c t s of c o l c h i c i n e . " Am. J . Anat., 88: 163 - 176. Roosen-Runge, E.C, G i e s e l , L.O., 1950 " Q u a n t i t a t i v e s t u d i e s on spermatogenesis i n the a l b i n o r a t . " Am. J . Anat., 87.: 1 - 30. Sapsford, C.S., 1962 "Changes i n the c e l l s of the sex chords and seminiferous tubules d u r i n g the development of the t e s t i s of the r a t and mouse." Aust. J . Zo o l . , 10: 1 7 8 - 1 9 3 . Sapsford, C.S., 1962 "The development of the t e s t i s of the Merino ram, w i t h s p e c i a l reference t o the o r i g i n of the a d u l t stem c e l l s . " Aust. J . A g r i c . Res., 13.: 487 - 502. 45 Smith, G., 1962 "The e f f e c t s of l i g a t i o n of the vasa e f f e r e n t i a and vasectomy on t e s t i c u l a r f u n c t i o n i n the a d u l t r a t . " J . E n d o c r i n o l . , 23_: 385 - 399. Smith, B.V.K., Lacy, D., 1959 "Residual bodies of seminiferous tubules of the r a t . " Nature, 184: 249 - 251. Steinberger, E., Dixon, W.J., 1959 "Some observations on the e f f e c t of heat on the t e s t i c u l a r germinal e p i t h -elium." F e r t i l . and S t e r i l . , 10: 578 - 595. Van Wagenen, G., 1926 "Degeneration and regeneration of the seminiferous tubules a f t e r l i g a t i o n of the d u c t u l i e f f e r e n t e s i n the r a t . " Anat. Rec. 32: 225. Van Wagenen, G., 1925 "Changes i n the t e s t i s of the r a t f o l l o w i n g l i g a t i o n of the d u c t u l i e f f e r e n t i . " Anat. R e c , 29: 399. Van Wagenen, G., 1924 "Degeneration of germinal e p i t h e l i u m i n the t e s t i s of the r a t as a r e s u l t of e f f e r e n t duct l i g a t i o n . " Anat. R e c , 27: 189. 4 6 KEY TO ABBREVIATIONS AC acre-some ASP a t r o p h i c spermatids BM basement membrane DS d i v i d i n g primary spermatocyte GC "ghost" c e l l HC head cap L seminiferous tubule lumen LC spermatids & c e l l u l a r d e b r i s i n lumen LS leptotene primary spermatocyte MF f u s i o n of nuclear membrane MNB m u l t i n u c l e a t e d body MNG m u l t i n u c l e a t e d "ghost" MS spermatid i n m i t o s i s NUC spermatid nucleus NN nu c l e o l u s PC pachytene primary spermatocyte PS d i p l o t e n e primary spermatocyte RB r e s i d u a l body RBM r e s i d u a l body mass RS r e s t i n g primary spermatocyte SC S e r t o l i c e l l nucleus SCC S e r t o l i c e l l cytoplasm SCS S e r t o l i c e l l s sloughing SG " spermatogonium SP spermatid SS secondary spermatocyte V vacuole VF vacuole forming ZS zygotene primary spermatocyte PLATES 47 PLATE 1 The Cycle of the Seminiferous E p i t h e l i u m i n the Mouse F i g . 2 - 1 3 . Representative seminiferous tubules from a nor-mal mouse t e s t i s showing the 12 stages of the c y c l e of the seminiferous e p i t h e l i u m . The c l a s s i f i c a t i o n of the c y c l e i s based on the f i r s t 12 steps of spermiogenesis. A l l 16 steps of spermiogenesis ( 1 - 3 , the G o l g i phase; 4 - 7 , the cap phase; 8 - 12, the acrosome phase; and 13 - 16, the matur-a t i o n phase) are i d e n t i f i e d by the changes i n the acrosomic system of spermatids as seen w i t h p e r i o d i c a c i d - S c h i f f . The haematoxylin step has been omitted from the s t a i n i n g proced-ure t o s i m p l i f y the changes i n the acrosomic system at each stage of the c y c l e . The c y c l e i s a continuous process of germ c e l l renewal, w i t h no beginning or end stages. The beginning of spermatogenesis, however, i s w i t h the mitoses of type A spermatogonia at stage IX of the c y c l e ( f i g . 10; t a b l e 2)• The nucleus and acrosome of stage IX spermatids undergo a marked e l o n g a t i o n , which continues through stage X l l ( f i g . 13). At l a t e stage X l l a new generation of spermatids ap-pears i n the germinal e p i t h e l i u m and marks the beginning of stage 1 of the c y c l e ( f i g . 2). The o l d e r generation of spermatids begin step 13 of spermiogenesis. The overlap of the two generations of spermatids p e r s i s t s through stages 1-V l l l . Stage 11 ( f i g . 3) i s i n i t i a t e d by the appearance of proacrosomic granules, which enlarge and fuse i n t o one l a r g e granule at stage 111 ( f i g . 4 ) . The head cap begins t o form at stage IV ( f i g . 5) and extends over the nucleus d u r i n g the next two stages. At stage V l l ( f i g . 8 ) , the head cap covers over o n e - t h i r d of the nucleus. The o l d e r generation of spermatids at the beginning of stage V l l enter step'16 of spermiogenesis - the l a s t morphological step of maturation. During stage V l l l ( f i g . 9) these mature spermatids are r e -leased i n t o the lumen of the t u b u l e , l e a v i n g only the one generation, step 8 spermatids i n the germinal e p i t h e l i u m . In f i g u r e 9, mature spermatids on the r i g h t s i d e of the tub-ul e have already been r e l e a s e d , but on the l e f t s i d e , most of the spermatids are s t i l l p resent. 725 X. 48 PLATE 2 F i g . 14. E l e c t r o n micrograph of a l o n g i t u d i n a l s e c t i o n of a spermatid from a c o n t r o l r a t t e s t i s showing the a n t e r i o r r e g i o n of the nucleus (NUC), the acrosome (AC), and the de-v e l o p i n g head cap (HC). The spermatid i s i n approximately step 8 of spermiogenesis. Compare the morphology of the head cap, which has been used as a c r i t e r i o n f o r the i d e n -t i f i c a t i o n of the stage of the seminiferous c y c l e , w i t h t h a t of step 8 spermatids prepared f o r l i g h t microscopy ( f i g . 9, and 57, 58). The surrounding S e r t o l i c e l l cytoplasm (SCC), separated from the spermatid only by the S e r t o l i plasma membrane, could p l a y an important r o l e not only i n the t r a n -sport of n u t r i e n t s from the basement membrane t o developing germ c e l l s , but a l s o i n the l o c a l r e g u l a t i o n of spermato-genesis. The t i s s u e was f i x e d w i t h glutaraldehyde and osmium t e x r o x i d e . Contrast was improved w i t h u r a n y l acetate and lead c i t r a t e . 45,000 X. 49 PLATES 3,4,5 & 6 The a t r o p h i c r e a c t i o n at each stage of the sem-i n i f e r o u s c y c l e i n the mouse, f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a i s represented on p l a t e s 3, 4, 5, and 6 ( f i g . 15 -86). The degenerative changes i l l u s t r a t e d are summed up i n t a b l e 6, page 17. The germinal e p i t h e l i u m i s o r i e n t a t e d i n a l l micrographs w i t h the basement membrane at the top and the lumen at the bottom. Roman numerals represent the 12 stages of the c y c l e . PAS - haematoxylin. 2000 X. a D A Y 5 D A Y "7 D A Y a D A Y 5 D A Y 7 D A Y 50 PLATE 7 Sections of seminiferous tubules f o l l o w i n g l i g a t i o n of the vasa e f f e r e n t i a , r e p r e s e n t a t i v e of the f i r s t 2 steps of tubule degener-a t i o n d u r i n g stages X - XIV of the seminiferous c y c l e i n the r a t . Iron haematoxylin. F i g . 87. Stage X l l of the seminiferous c y c l e from a c o n t r o l r a t t e s t i s . The germinal e p i t h e l i u m c o n s i s t s of S e r t o l i c e l l n u c l e i (SC) and type A spermatogonia (SG) adjacent t o the basement membrane; zygotene (ZS), then pachytene (PC) primary spermatocytes; and spermatids (SP) i n step 12 of spermiogenesis beside the lumen. 1350 X. F i g . 88. Approximately stage X l l of the seminiferous c y c l e d u r i n g the f i r s t step i n seminiferous tubule degeneration -the " c e l l u l a r sloughing" stage. Spermatids have been pre-maturely r e l e a s e d , and s e v e r a l pachytene primary spermato-cytes (PC) are i n the process of being sloughed i n t o the lumen ( L ) . Most zygotene (ZS), and some pachytene primary spermatocytes i n the germinal e p i t h e l i u m s t i l l appear mor-p h o l o g i c a l l y normal. Some S e r t o l i c e l l n u c l e i (SC) have migrated away from the basement membrane. 1250 X. F i g . 89 & 90. Regions of 2 d i f f e r e n t seminiferous tubules i n the "spermatocyte ghost c e l l " stage of tubule degener-a t i o n . In f i g . 89, the number of pachytene primary sper-matocytes i s reduced as a r e s u l t of c e l l u l a r sloughing and degeneration w i t h i n the germinal e p i t h e l i u m . Two pachytene primary spermatocytes w i t h p y k n o t i c n u c l e i have been sloughed i n t o the lumen; the remainder have e i t h e r p y k n o t i c n u c l e i or have undergone chromatolysis and appear as "ghost" c e l l s (GC) w i t h l i t t l e or no nuclear m a t e r i a l and f a i n t PAS - p o s i t i v e cytoplasm. Most zygotene spermatocytes s t i l l appear morphol-o g i c a l l y normal. The cytoplasm of the S e r t o l i c e l l s (SCC) i s much more conspicuous and contains numerous vacuoles (V). The S e r t o l i c e l l n u c l e i have increased i n number, and a few are l o c a t e d away from the basement membrane. Towards the end of the "spermatocyte ghost c e l l " stage ( f i g . 90), as the degen-e r a t i o n of the germinal e p i t h e l i u m continues, the number of spermatocytes i s g r e a t l y depleted. Numerous "ghost" c e l l s (GC) of degenerating pachytene and zygotene spermatocytes are p r e s e n t . Vacuoles (V) i n the S e r t o l i c e l l cytoplasm are abundant 1250 X 51 PLATE 8 Sections of seminiferous tubules r e p r e s e n t -a t i v e of the steps of tubule degeneration d u r i n g stages 1 - IX of the seminiferous c y c l e i n the r a t . Iron Haematoxylin. F i g . 91. Stage 111 of the seminiferous c y c l e d u r i n g the "spermatid sloughing" stage. Most of step 16 spermatids are s t i l l present i n the germinal e p i t h e l i u m . A few step 3 spermatids are about t o be sloughed. Intermediate spermatogonia, pachytene primary spermatocytes and S e r t o l i c e l l n u c l e i are m o r p h o l o g i c a l l y normal. 1450 X, F i g . 92, Approximately stage V l l of the c y c l e . During the l a t t e r p a r t of the "spermatid sloughing" stage of tubule degeneration. The younger generation of spermatids have undergone py k n o t i c changes, producing nuclear r i n g forma-t i o n s of chromatin (ASP). The mature spermatids have a l l been e x f o l i a t e d . A few S e r t o l i c e l l n u c l e i have migrated from the basement membrane. 1250 X. F i g . 93. Approximately stage V of the seminiferous c y c l e at the beginning of the "multinucleated body" stage of tubule degeneration. M u l t i n u c l e a t e d bodies of spermatids (MNB) have formed as a r e s u l t of f u s i o n of nuclear mem-branes of adjacent n e c r o t i c spermatids (ASP), The S e r t o l i c e l l cytoplasm (SCC) becomes much more conspicuous and appears t o expand t o f i l l much of the lumen ( L ) . S e r t o l i c e l l n u c l e i (SC) i n c r e a s e i n number and migrate away from the basement membrane. 1250 X. F i g . 94. The f u s i o n of 2 almost m o r p h o l o g i c a l l y normal spermatid n u c l e i at steps 4 - 5 i n spermiogenesis i n the mouse. The p o i n t of f u s i o n i s at the opposite end t o the head cap, but f u s i o n of spermatid n u c l e i i s g e n e r a l l y not r e s t r i c t e d t o any one p o i n t . PAS - haematoxylin. 10,300 X. 52 PLATE 9 F i g . 95. A l a t e r step of the "multinucleated body" stage •-'of tubule degeneration than f i g u r e 94. Most of the n e c r o t i c spermatid n u c l e i have undergone membrane f u s i o n w i t h ad-jac e n t spermatid n u c l e i , forming l a r g e r m u l t i n u c l e a t e d bodies (MNB). Note t h a t a l l the MNB are lo c a t e d i n the germinal e p i t h e l i u m . Iron haematoxylin. 1250 X. F i g . 96. A d e v i a t i o n from the u s u a l form of tubule degener-a t i o n d u r i n g stages 1 - IX of the c y c l e . The germinal e p i t h -elium i s g r e a t l y reduced i n s i z e and the lumen (L) i s com-p l e t e l y blocked w i t h germ c e l l s and c e l l u l a r d e b r i s (LC). MNB are found i n the lumen as w e l l as i n the germinal e p i t h e -l i u m . The S e r t o l i c e l l n u c l e i have increased i n number. Note the accumulation of S e r t o l i c e l l n u c l e i at a break i n the basement membrane at the upper r i g h t corner. Iron haematox-y l i n . 1250 X. F i g . 97. The end of the "multinucleated body" stage i n de-generation i s marked w i t h only a few MNB remaining. The S e r t o l i c e l l cytoplasm (SCC) i s r e l a t i v e l y c l e a r of degener-a t i n g germ c e l l s and contains numerous vacuoles (V). Iron haematoxylin. * 1200 X. F i g . 98. A s e c t i o n t a n g e n t i a l t o the lumen (L) duri n g the " S e r t o l i c e l l cytoplasm" stage i n tubule degeneration. The germinal e p i t h e l i u m c o n s i s t s of a few pachytene primary spermatocytes, spermatogonia and numerous S e r t o l i c e l l n u c l e i . The t a n g e n t i a l s e c t i o n i s an advantageous s e c t i o n t o i l l u s -t r a t e the m i g r a t i o n of S e r t o l i c e l l n u c l e i away from the base-ment membrane. Iron haematoxylin, 1200 X. 53 PLATE 10 Seminiferous tubules i n the " S e r t o l i c e l l s loughing" stage of seminiferous tubule de generation d u r i n g severe atrophy. F i g . 99. L o n g i t u d i n a l s e c t i o n of seminiferous tubules from a r a t t e s t i s . The lower tubule has undergone severe S e r t o l i c e l l sloughing, r e s u l t i n g i n a t h i n germinal e p i t h e l i u m con-s i s t i n g of S e r t o l i c e l l s , spermatogonia and a few primary spermatocytes up t o the pachytene maturation stage. A mass of germinal e p i t h e l i u m c o n t a i n i n g numerous S e r t o l i c e l l s (SCS) i s about t o be sloughed. Iron haematoxylin. 550 X. F i g . 100. A higher m a g n i f i c a t i o n of the S e r t o l i c e l l sloughing c o n d i t i o n i n d i c a t e d i n f i g u r e 99 (SCS), but taken from mouse t e s t i s . Numerous S e r t o l i c e l l n u c l e i (SC) and o c c a s i o n a l pachytene primary spermatocytes (PC) are being sloughed i n t o the lumen. PAS - haematoxylin. 2400 X. F i g . 101. A group of S e r t o l i c e l l s t h a t have been sloughed i n t o the lumen. The n u c l e i s t i l l appear m o r p h o l o g i c a l l y normal. A p o s s i b l e p y k n o t i c pachytene spermatocyte i s i n c l u d e d i n the S e r t o l i c e l l cytoplasmic mass. PAS - haematoxylin. 3600 X. 54 PLATE 11 The v a r i a b i l i t y i n the r a t e of seminiferous tubule degeneration i n r a t t e s t e s t h a t have been subject t o l i g a t i o n of the vasa e f f e r -e n t i a f o r 4 days. Iron haematoxylin. F i g . 102. Various degrees of tubule atrophy i n d i f f e r e n t t u b u l e s . The degenerative changes make i t d i f f i c u l t t o a c c u r a t e l y determine the stage of the c y c l e . The stages l i s t e d below are t h e r e f o r e approximations. 500 X. Tubule B: early, "multinucleated body" stage of tubule degeneration d u r i n g stage 111 of the c y c l e . Tubule A: more advanced "multinucleated body" stage of degeneration d u r i n g stage V of the c y c l e . Tubule B±i e a r l y "spermatocyte ghost c e l l " stage of degeneration d u r i n g stage X of the c y c l e . Tubule C: advanced " S e r t o l i c e l l sloughing" stage of degeneration at stage V l l of the c y c l e . F i g . 103. An extremely a t r o p h i c seminiferous tubule (B, Bx, B 2 ) adjacent t o a tubule i n only a s l i g h t degenerative con-d i t i o n (A, A J L , A 2 ) . Note t h a t although the r a t e of atrophy v a r i e s i n d i f f e r e n t t u b u l e s , the degree of degeneration g e n e r a l l y remains the same throughout the length of the tubule (eg. tubule B; and i n f i g u r e 102, tubules A and C). 500 X. 55 PLATE 12 The f a t e of m u l t i n u c l e a t e d bodies d u r i n g the "multinucleated body" stage of seminiferous tubule degeneration stages 1 - IX of the c y c l e i n the r a t t e s t i s . Iron haematoxylin. F i g . 104. Stage VI: the c e l l s l i n i n g the tubule are type B spermatogonia (SG), S e r t o l i c e l l n u c l e i (SC), and a few pachytene primary spermatocytes (PC). Spermatids have form-ed m u l t i n u c l e a t e d bodies (MNB) i n v a r i o u s s t a t e s of degener-a t i o n . The MNB c o n s i s t s of p e r i p h e r a l l y l o c a t e d spermatid n u c l e i and a c e n t r a l cytoplasmic mass. Three stages of MNB degeneration can be d i f f e r e n t i a t e d : Stage 1 MNB c o n t a i n the most hyperchromatic n u c l e i . Stage 2 MNB show a marked r e d u c t i o n i n the i n t e n s i t y of the nuclear and cytoplasmic s t a i n i n g . Stage 3 MNB c o n t a i n spermatids w i t h d i s r u p t e d nuclear membranes, and have been termed " m u l t i -nucleated ghosts" (MNG). S e r t o l i c e l l n u c l e i (SC) are l o c a t e d c l o s e to the MNB and o f t e n conform t o the shape of the edge of a MNB. 1350 X. F i g . 105. Approximately stage V l l of the c y c l e . Stages 1 and 2 MNB are present, and two l a t e stage 3 MNG - which have become g r e a t l y d i s o r g a n i z e d . One MNG has been e x f o l i a t e d i n t o the tubule lumen ( L ) . Stage 4 MNG i n v o l v e s the l y s i s of the remaining c e l l u l a r mass. The disappearance of the MNG i s concurrent w i t h the formation of vacuoles w i t h i n the germinal e p i t h e l i u m . 1350 X. 56 PLATE 13 The f a t e of r e s i d u a l bodies d u r i n g t e s t i c -u l a r atrophy i n the r a t . Iron haematoxylin. F i g . 106. L o n g i t u d i n a l s e c t i o n of a r e g i o n of a normal sem-i n i f e r o u s tubule i n stage V l l . The l a r g e b a s o p h i l i c bodies i n the lumen (L) and at the edge of the germinal e p i t h e l i u m are cytoplasmic r e s i d u a l bodies (RB) r e l e a s e d by step 19 spermatids. 1250 X. F i g . 107. L o n g i t u d i n a l s e c t i o n of a r e g i o n of an a t r o p h i c seminiferous tubule i n stage V l l . R e s idual bodies have been phagocytized by S e r t o l i c e l l s and are being gathered i n t o residual'body masses (RBM). M u l t i n u c l e a t e d bodies (MNB) of step 7 spermatids can be seen i n v a r i o u s s t a t e s of degeneration. S e r t o l i c e l l n u c l e i (SC) have increased i n number, and some have migrated away from the basement mem-brane. 1250 X. F i g . 108. Stage V l l : most r e s i d u a l bodies have been organ-i z e d i n t o r e s i d u a l body masses and resemble the m u l t i n u c -l e a t e d bodies of spermatids. R e s t i n g primary spermatocytes (RS), S e r t o l i c e l l n u c l e i and a few pachytene primary sper-matocytes l i n e the basement membrane. Numerous vacuoles (V) are present w i t h i n the S e r t o l i c e l l cytoplasm (SCC). 1250 X. F i g . 109. Cross s e c t i o n of a seminiferous tubule s i m i l a r t o t h a t seen i n f i g u r e 108. Note the r e s i d u a l bodies are l o c a -ted only i n the r e s i d u a l body masses w i t h i n the cytoplasm of S e r t o l i c e l l s . 1200 X. 57 PLATE 14 L i m i t e d atrophy of a seminiferous tubule i n a c o n t r o l t e s t i s of a mouse. PAS- haemat-o x y l i n • F i g . 110. Stage 1 of the seminiferous c y c l e : most of the tubule appears normal, but at the bottom of the t u b u l e , both generations of spermatids (SP) have undergone degen-e r a t i v e changes. Step 13 spermatids have been sloughed i n t o the lumen, and some of the step 1 spermatids have formed m u l t i n u c l e a t e d bodies (MNB). 1300 X. F i g . 111. Stage X l l of the c y c l e : only a s m a l l p o r t i o n of the tubule appears normal. In the a t r o p h i c r e g i o n of the t u b u l e , step 12 spermatids have a l l been sloughed, and some secondary spermatocytes are i n the process of being sloughed. M u l t i n u c l e a t e d bodies (MNB) of secondary sper-matocytes have formed i n a s i m i l a r manner as MNB of sper-matids. Some d i p l o t e n e primary spermatocytes (PS) are present which have not yet d i v i d e d t o form secondary spermatocytes. 1300 X. F i g . 112. Higher m a g n i f i c a t i o n of two secondary spermato-cytes whose nuclear membranes have fused together. 2600 X. 58 PLATE 15 Changes i n the contents of the epididymal duct f o l l o w i n g l i g a t i o n of the vasa e f f e r -e n t i a i n mice* PAS - haematoxylin. 340 X. F i g . 113. S e c t i o n of the caput e p i d i d y m i d i s from a c o n t r o l t e s t i s . Every duct s e c t i o n contains spermatozoa. F i g . 114. At 1 day p o s t l i g a t i o n , the contents of the e p i d -idymal duct show no apparent change. F i g . 115. At 2 days p o s t l i g a t i o n , the number of spermatozoa i n the duct have decreased s l i g h t l y . F i g . 116. At 4 days p o s t l i g a t i o n , some s e c t i o n s of the duct are almost devoid of spermatozoa. The r e g i o n of the duct t h a t has been cut l o n g i t u d i n a l l y contains d i s o r g a n i z e d areas of n e c r o t i c spermatozoa and PAS - p o s i t i v e amorphous masses of mucus. F i g . 117. At 5 days p o s t l i g a t i o n , the contents of a l l duct s e c t i o n s are abnormal. Many regions of the duct are devoid of spermatozoa. F i g . 118. At 7 days p o s t l i g a t i o n , most regions of the duct are devoid of spermatozoa or c o n t a i n s m a l l clumps of ne-c r o t i c spermatozoa and mucus. 

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