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Motile components in early rat spermatids
Cell Differentiation, 10 (1981) 157--161
157
Elsevier/North-Holland Biomedical Press
MOTILE COMPONENTS IN E A R L Y R A T SPERMATIDS H. WALT Institute of Pathology, University of Ziirich, University Hospital, CH-8091 Ziirich, Switzerland
(Accepted 30 January 1981)
In early rat spermatids, two distinctly different kinds of movements of cell components were detected by video-analysis. The primary flagellum, a typical 9 + 2 axonema, is capable of inducing wave-like movements in three dimensions, unlike late spermatid forms, which display motility of the now thickened flagellum only by repeated bending of its extreme part. Additionally, at the apical regions of spermatids of the same early stage, cytoplasmic protrusions executed rhythmic movements at a frequency of almost three times per second. The two kinds of motility of the different components in the same cell type are thought to be involved in normal orientation and transfer of spermatids in the tubulus seminiferus during their differentiation to sperm. Germ cell transport
spermatid
motile flagellum
1. Introduction T ran s p o r t o f essentially immotile sperm along the seminiferous tubules to the rete testis is achieved by the following components: contractility of the testicular capsule and o f the seminiferous tubules, and flow of tubular fluid (Ellis et al., 1978). During their differentiation to sperm, germ cells are transferred within the germinal epithelium of the tubular wall to the tubular lumen. Over this short distance, germ cells undergo remarkable processes, namely mitosis (spermatogonia), meiosis (spermatocytes) and spermiogenesis (spermatids), leading finally to highly differentiated sperm cells. Differentiation of germ cells is influenced partly by Sertoli cells, which are elements of somatic origin. T h e y fulfill different functions, such as nutrition and support o f germ cells, as well as resorption o f spermatid cytoplasm. Sertoli cells are t h o u g h t to be responsible alone for the transp o r t o f differentiating germ cells in the direction o f the tubular lumen and for release of mature sperm ( F a w c e t t and Phillips, 1969; Setchell, 1978; Russell, 1979, 1980). Re-
cytoplasmic movement
cently, it was possible to demonstrate active motility o f the primary spermatid flagellum in rat and also in man (Walt, 1980). The present work examines the motions of rat spermatid c o m p o n e n t s in greater detail. The findings reveal two distinctly different kinds of motile c o m p o n e n t s in spermatids of the same stage.
2. Animals, material and m e t h o d s In this study, we used healthy, 4-month old male albino rats (Iva: SIV, Ivanovas, Kisslegg, Germany), 350--400 g in weight. T h e y were kept in groups of 3--4 per cage in our central animal facilities at constant t em perat ure (21°C) and humidity (55% r.h.), fed on pelleted standard diet and water ad libitum. T he animals to be killed were first anesthesized with ether, and then given an overdose of intraperitoneally injected Inactin (Byk Gulden, Konstanz, Germany). The testes were removed and submerged in ice-cold Ringer's solution (8.6 g NaCI, 0.33 g CaC12, 0.3 g KC1 per liter aqua bidest., pH 7.2). The tunica albuginea was stripped off carefully, and short
0045-6039/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
158 pieces o f single tubuli seminiferi were placed in a d r o p o f Ringer's s o l u t i o n on a m i c r o s c o p e slide. The tubules were f r a g m e n t e d mechanically with fine forceps, covered b y coverslips and a n a l y z e d in a light m i c r o s c o p e (phasec o n t r a s t ) , e q u i p p e d with a c o l o r T V - c a m e r a plus S o n y - v i d e o r e c o r d i n g system. S t o p f r a m e pictures f r o m t h e video tape were p h o t o g r a p h e d directly f r o m the T V - m o n i t o r .
3. Results The following germ cells in d i f f e r e n t stages of d e v e l o p m e n t c o u l d be identified b y phase c o n t r a s t m i c r o s c o p y : s p e r m a t o g o n i a , spermat o c y t e s , spermatids and sperm. T h e last t w o t y p e s are d e t e c t a b l e at low magnification. S p e r m s are e n d o w e d with a h o o k e d head and a thick flagellar tail and are immotile. Early
Fig. 1. Early rat spermatid with its primary flagellum in wave-like and three-dimensional movement. Time between phases a, b and c: 0.4 s.
Fig. 2. Late rat spermatid with the characteristic spindle-shaped body in the middle part of the flagellum. Its movement is restricted to bending of thee extreme part of the flagellum. The points represent the distance passed by the flagellar end. The time between phases a and b: 0.3 s. One early spermatid with two primary flagella (arrows) in motion are detectable as well. S = spindle-shaped body; H = spermatid head with attached cytoplasmic fragments.
159
Fig. 3. Four typical subsequent phases (a--d) of movement of the apically localized spermatid protrusion. In a, the spermatid gives a round aspect and no protrusion is detectable. The protrusion b e c o m e s visible in b, increases progressively (c) and diminishes after (d) achieving the form of (a) again. Time difference between each phase: 0.4 s. N = nucleus.
spermatids are round to oval and contain a thin primary flagellum, the axonema. Later spermatids are equipped with a thicker flagellum and a spindle-shaped body, localized in the middle portion of the flagellum. At this stage of differentiation most of the cytoplasm has become resorbed. Both types of spermatids demonstrate two different forms of flagellar motility. The axonema of early spermatids is capable of inducing wave-like motions in three dimensions along the whole flagellum {Fig. 1). The later forms, n o w containing a thickened flagellum, are mainly motile near the end portion of the flagellum (Fig. 2). This movement, however, is limited to repeated two-dimensional bending of the extreme part of that flagellar portion. Surprisingly, early spermatids often display another, separate form of cellular motion. Cytoplasm protrudes especially at the apical region of the spermatid and begins to execute rhythmic movements at a frequency of 2.5 times/s, for at least 2 min (Fig. 3). The movement then slows progressively until motion ends and the protrusion disappears. The spermatid nuclei seem to be localized in the center of the cell during all of these phases, corresponding to Stages III--VII (Leblond and Clermont, 1952).
4. Discussion In the literature, the analysis of germ cell motion in seminiferous tubules of various mammals is limited to the description of sperm motility. There is agreement, supported by our findings, that testicular sperm is essentially immotile (Yochem, 1930; Voglmayr et al., 1967; Setchell et al., 1969). Nevertheless, the results of the present study have shown that rat testicular spermatids possess two motile components: the primary flagellum is capable of motion, and spermatids of the same stage of development may protrude parts of their cytoplasm which display striking movements (Fig. 4). Both forms of cellular motions may be of importance in the normal transfer of these cells within the germinal epithelium and in transport of sperm. Recently, gap junctions
C
p
Fig. 4. S c h e m a t i c drawing o f an early rat spermatid, indicating m o v e m e n t s o f the flagellum (F) and the protrusion (P) at the apical cell area. Three subseq u e n t phases are m a r k e d as follows: 1) , 2) . . . . . . , and 3) . . . . . . . The 0-phase b e t w e e n the phases 3 and 1, representing a round cell w i t h o u t protrusion, is n o t indicated. N = nucleus; C = cytoplasm.
160 in rat could only be detected between Sertoli cells and germ cells of earlier phases, i.e. spermatocytes and spermatogonia (McGinley et al., 1979). These authors could n o t find similar junctions between Sertoli cells and spermarids. On the other hand, it could be shown by experiment that in areas of contact between Sertoli cells and spermatids Sertoli cell junctional specializations are developed, acting as cohesive devices which can resist mechanical stress (Romrell and Ross, 1979). These junctions appear at Stage VIII and later, and are localized precisely where the developing acrosome makes contact with the spermatid membrane. At earlier stages, no similar cell junctions could be found by freeze fracture between spermatids and Sertoli cells, but were detected on thin sections. In addition, adjacent spermatids are connected by intercellular bridges {Gondos, 1973). In the present work, whole viable spermatids were analyzed, but numerical counts and precise histologic staging were n o t done. From inspecting intact pieces of germinal epithelium, however, we believe that all spermatids are capable of performing the abovementioned motions, but that a small number might be damaged during preparation. At the time of movement analysis, the cell nucleus was localized in the center of the round to oval spermatid, i.e. the findings concern earlier spermatid stages (Stages III--VII). During these phases, the spermatid takes up its definite position within the germinal epithelium, partially by cell rotation. Spermatids then keep their position relative to the germinal epithelium with the flagellum pointing to the tubular lumen, the head to the base of the germinal epithelium. Since this rotation occurs at about the same stage as the movement of the apical spermatid region, one may speculate that the two processes are causally related. Several possibilities may explain the basic mechanism responsible for the astonishing cytoplasmic movements of spermatids. In the electron microscope, a so-called manchette was detected in early spermatids of various
mammalian species, including man. It consists of numerous microtubuli which circle the spermatid nucleus radiating to the posterior cytoplasmic area. The function of the manchette still remains obscure. It has been postulated that this apparatus is involved in convecting the cytoplasm within the spermatid (Fawcett et al., 1971). As a result of change of intracytoplasmic conditions, the cell membrane may allow the cytoplasm to protrude in definite places, where the above-mentioned motions then occur. Another possible mechanism may well include the presence of contractile elements already in spermatids. In the testicular sperm of the rat, actin has been demonstrated in the subacrosomal region (Campanella et al., 1979). The same authors found actin and myosin in human sperm and suggest that myosin may possibly be present in rat sperm as well. Whether microtubules or early muscle proteins or both are responsible for the protrusions and the rhythmic motions still remains unclear. The second motile element found in spermatids, namely the axonema, could be responsible for several effects. This typical 9 + 2 axonema is obviously motile immediately after differentiation. Spermatids from Stage VIII and the following stages, integrated in the germinal epithelium, are arranged such that their flagella point to the tubular lumen and the heads to the basement membrane as mentioned above. Recently, I found that free spermatids are propelled by their flagella pushing from behind (Walt, 1980). If, in fact, the flagella of primary spermatids were to behave in the same manner in intact tubules, the flagella of the fixed spermatids would tend to displace their surroundings in the direction of the tubular lumen. Consequently, tubular fluid, sperm and probably even immotile late spermatids, still somewhat fixed to a Sertoli cell by the tubulobular complex (Russell and Clermont, 1976), could be transferred by the resultant mechanical force into the center of the lumen, where the mass of sperm is present. In addition, this spermatic flux
161 m a y be aided t o f l o w m o r e easily along t h e seminiferous tubules through the influence of m o t i l e s p e r m a t i d flagella, f o r m i n g s o m e k i n d o f a flagellated cell layer. T h u s , t h e p h a s e b e t w e e n s p e r m a t i d differe n t i a t i o n a n d s p e r m release seems t o be d o m i n a t e d n o t o n l y b y t h e Sertoli cell as t h e active m e c h a n i s m in m o v i n g of d i f f e r e n t i a t i n g g e r m cells. T h e striking m o t i o n s o f early sperm a t i d c o m p o n e n t s suggest t h a t t h e s e cells d o themselves contribute to their orientation and t r a n s f e r w i t h i n t h e germinal e p i t h e l i u m t o t h e l i b e r a t i o n a n d t r a n s p o r t o f t h e i r m a t u r e stages.
Acknowledgements T h e a u t h o r w o u l d like t o t h a n k Mrs. M. Stillhart ( D e p a r t m e n t o f N e p h r o l o g y ) , Mr. J. B e r w e r t a n d Mr. B. L o r e n z e t t i ( U n i v e r s i t y T V - T e a m ) , Miss B. Stahel, Mr. Ch. H~iberlin and Mr. H. N e f o f o u r I n s t i t u t e f o r t h e i r v a l u a b l e assistance, a n d Prof. Chr. H e d i n g e r and m y colleague Dr. A. y o n H o c h s t e t t e r f o r t h e i r critical c o m m e n t s .
References Campanella, C., G. Gabbiani, B. Baccetti, A.G. Burrini and V. Pallini: J. Submicrosc. Cytol. 11, 53-71 (1979). Ellis, L.C., J.R. Buhrley and J.L. Hargrove: Arch. Androl. 1,139--146 (1978). Fawcett, D.W., W. Anderson and D.M. Phillips: Dev. Biol. 26, 220--251 (1971). Fawcett, D.W. and D.M. Phillips: J. Reprod. Fertil. Suppl..6,405--418 (1969). Gondos, B.: Differentiation 1,177--182 (1973). Leblond~ C.P. and Y. Clermont: Ann. N.Y. Acad. Sci. 55, 548--573 (1952). McGinley, D.M., Z. Posalaky, M. Provaznik and L.D. Russell: Tissue Cell 11,741--754 (1979). Romrell, L.J. and M.H. Ross: Anat. Rec. 193, 23--42 (1979). Russell, L.D.: Anat. Rec. 194, 213--232 (1979). Russell, L.D.: Gamete Res. 3,179--202 (1980). Russell, L.D. and Y. Clermont: Anat. Rec. 185, 259-278 (1976). Setchell, B.P.: In: The Mammalian Testis. Elek Books Limited, London (1978). Setchell, B.P., T.W. Scott, J.K. Voglmayr and G.M.H. Waites: Biol. Reprod. Suppl. 1, 40--66 (1969). Voglmayr, J.K., T.W. Scott, B.P. Setchell and G.M.H. Waites: J. Reprod. Fertil. 14, 87--99 (1967). Walt, H.: Eur. J. Cell Biol. 22,559 (1980). Yochem, D.E.: Physiol. Zool. 3,309--329 (1930).
157
Elsevier/North-Holland Biomedical Press
MOTILE COMPONENTS IN E A R L Y R A T SPERMATIDS H. WALT Institute of Pathology, University of Ziirich, University Hospital, CH-8091 Ziirich, Switzerland
(Accepted 30 January 1981)
In early rat spermatids, two distinctly different kinds of movements of cell components were detected by video-analysis. The primary flagellum, a typical 9 + 2 axonema, is capable of inducing wave-like movements in three dimensions, unlike late spermatid forms, which display motility of the now thickened flagellum only by repeated bending of its extreme part. Additionally, at the apical regions of spermatids of the same early stage, cytoplasmic protrusions executed rhythmic movements at a frequency of almost three times per second. The two kinds of motility of the different components in the same cell type are thought to be involved in normal orientation and transfer of spermatids in the tubulus seminiferus during their differentiation to sperm. Germ cell transport
spermatid
motile flagellum
1. Introduction T ran s p o r t o f essentially immotile sperm along the seminiferous tubules to the rete testis is achieved by the following components: contractility of the testicular capsule and o f the seminiferous tubules, and flow of tubular fluid (Ellis et al., 1978). During their differentiation to sperm, germ cells are transferred within the germinal epithelium of the tubular wall to the tubular lumen. Over this short distance, germ cells undergo remarkable processes, namely mitosis (spermatogonia), meiosis (spermatocytes) and spermiogenesis (spermatids), leading finally to highly differentiated sperm cells. Differentiation of germ cells is influenced partly by Sertoli cells, which are elements of somatic origin. T h e y fulfill different functions, such as nutrition and support o f germ cells, as well as resorption o f spermatid cytoplasm. Sertoli cells are t h o u g h t to be responsible alone for the transp o r t o f differentiating germ cells in the direction o f the tubular lumen and for release of mature sperm ( F a w c e t t and Phillips, 1969; Setchell, 1978; Russell, 1979, 1980). Re-
cytoplasmic movement
cently, it was possible to demonstrate active motility o f the primary spermatid flagellum in rat and also in man (Walt, 1980). The present work examines the motions of rat spermatid c o m p o n e n t s in greater detail. The findings reveal two distinctly different kinds of motile c o m p o n e n t s in spermatids of the same stage.
2. Animals, material and m e t h o d s In this study, we used healthy, 4-month old male albino rats (Iva: SIV, Ivanovas, Kisslegg, Germany), 350--400 g in weight. T h e y were kept in groups of 3--4 per cage in our central animal facilities at constant t em perat ure (21°C) and humidity (55% r.h.), fed on pelleted standard diet and water ad libitum. T he animals to be killed were first anesthesized with ether, and then given an overdose of intraperitoneally injected Inactin (Byk Gulden, Konstanz, Germany). The testes were removed and submerged in ice-cold Ringer's solution (8.6 g NaCI, 0.33 g CaC12, 0.3 g KC1 per liter aqua bidest., pH 7.2). The tunica albuginea was stripped off carefully, and short
0045-6039/81/0000--0000/$02.50 © Elsevier/North-Holland Biomedical Press
158 pieces o f single tubuli seminiferi were placed in a d r o p o f Ringer's s o l u t i o n on a m i c r o s c o p e slide. The tubules were f r a g m e n t e d mechanically with fine forceps, covered b y coverslips and a n a l y z e d in a light m i c r o s c o p e (phasec o n t r a s t ) , e q u i p p e d with a c o l o r T V - c a m e r a plus S o n y - v i d e o r e c o r d i n g system. S t o p f r a m e pictures f r o m t h e video tape were p h o t o g r a p h e d directly f r o m the T V - m o n i t o r .
3. Results The following germ cells in d i f f e r e n t stages of d e v e l o p m e n t c o u l d be identified b y phase c o n t r a s t m i c r o s c o p y : s p e r m a t o g o n i a , spermat o c y t e s , spermatids and sperm. T h e last t w o t y p e s are d e t e c t a b l e at low magnification. S p e r m s are e n d o w e d with a h o o k e d head and a thick flagellar tail and are immotile. Early
Fig. 1. Early rat spermatid with its primary flagellum in wave-like and three-dimensional movement. Time between phases a, b and c: 0.4 s.
Fig. 2. Late rat spermatid with the characteristic spindle-shaped body in the middle part of the flagellum. Its movement is restricted to bending of thee extreme part of the flagellum. The points represent the distance passed by the flagellar end. The time between phases a and b: 0.3 s. One early spermatid with two primary flagella (arrows) in motion are detectable as well. S = spindle-shaped body; H = spermatid head with attached cytoplasmic fragments.
159
Fig. 3. Four typical subsequent phases (a--d) of movement of the apically localized spermatid protrusion. In a, the spermatid gives a round aspect and no protrusion is detectable. The protrusion b e c o m e s visible in b, increases progressively (c) and diminishes after (d) achieving the form of (a) again. Time difference between each phase: 0.4 s. N = nucleus.
spermatids are round to oval and contain a thin primary flagellum, the axonema. Later spermatids are equipped with a thicker flagellum and a spindle-shaped body, localized in the middle portion of the flagellum. At this stage of differentiation most of the cytoplasm has become resorbed. Both types of spermatids demonstrate two different forms of flagellar motility. The axonema of early spermatids is capable of inducing wave-like motions in three dimensions along the whole flagellum {Fig. 1). The later forms, n o w containing a thickened flagellum, are mainly motile near the end portion of the flagellum (Fig. 2). This movement, however, is limited to repeated two-dimensional bending of the extreme part of that flagellar portion. Surprisingly, early spermatids often display another, separate form of cellular motion. Cytoplasm protrudes especially at the apical region of the spermatid and begins to execute rhythmic movements at a frequency of 2.5 times/s, for at least 2 min (Fig. 3). The movement then slows progressively until motion ends and the protrusion disappears. The spermatid nuclei seem to be localized in the center of the cell during all of these phases, corresponding to Stages III--VII (Leblond and Clermont, 1952).
4. Discussion In the literature, the analysis of germ cell motion in seminiferous tubules of various mammals is limited to the description of sperm motility. There is agreement, supported by our findings, that testicular sperm is essentially immotile (Yochem, 1930; Voglmayr et al., 1967; Setchell et al., 1969). Nevertheless, the results of the present study have shown that rat testicular spermatids possess two motile components: the primary flagellum is capable of motion, and spermatids of the same stage of development may protrude parts of their cytoplasm which display striking movements (Fig. 4). Both forms of cellular motions may be of importance in the normal transfer of these cells within the germinal epithelium and in transport of sperm. Recently, gap junctions
C
p
Fig. 4. S c h e m a t i c drawing o f an early rat spermatid, indicating m o v e m e n t s o f the flagellum (F) and the protrusion (P) at the apical cell area. Three subseq u e n t phases are m a r k e d as follows: 1) , 2) . . . . . . , and 3) . . . . . . . The 0-phase b e t w e e n the phases 3 and 1, representing a round cell w i t h o u t protrusion, is n o t indicated. N = nucleus; C = cytoplasm.
160 in rat could only be detected between Sertoli cells and germ cells of earlier phases, i.e. spermatocytes and spermatogonia (McGinley et al., 1979). These authors could n o t find similar junctions between Sertoli cells and spermarids. On the other hand, it could be shown by experiment that in areas of contact between Sertoli cells and spermatids Sertoli cell junctional specializations are developed, acting as cohesive devices which can resist mechanical stress (Romrell and Ross, 1979). These junctions appear at Stage VIII and later, and are localized precisely where the developing acrosome makes contact with the spermatid membrane. At earlier stages, no similar cell junctions could be found by freeze fracture between spermatids and Sertoli cells, but were detected on thin sections. In addition, adjacent spermatids are connected by intercellular bridges {Gondos, 1973). In the present work, whole viable spermatids were analyzed, but numerical counts and precise histologic staging were n o t done. From inspecting intact pieces of germinal epithelium, however, we believe that all spermatids are capable of performing the abovementioned motions, but that a small number might be damaged during preparation. At the time of movement analysis, the cell nucleus was localized in the center of the round to oval spermatid, i.e. the findings concern earlier spermatid stages (Stages III--VII). During these phases, the spermatid takes up its definite position within the germinal epithelium, partially by cell rotation. Spermatids then keep their position relative to the germinal epithelium with the flagellum pointing to the tubular lumen, the head to the base of the germinal epithelium. Since this rotation occurs at about the same stage as the movement of the apical spermatid region, one may speculate that the two processes are causally related. Several possibilities may explain the basic mechanism responsible for the astonishing cytoplasmic movements of spermatids. In the electron microscope, a so-called manchette was detected in early spermatids of various
mammalian species, including man. It consists of numerous microtubuli which circle the spermatid nucleus radiating to the posterior cytoplasmic area. The function of the manchette still remains obscure. It has been postulated that this apparatus is involved in convecting the cytoplasm within the spermatid (Fawcett et al., 1971). As a result of change of intracytoplasmic conditions, the cell membrane may allow the cytoplasm to protrude in definite places, where the above-mentioned motions then occur. Another possible mechanism may well include the presence of contractile elements already in spermatids. In the testicular sperm of the rat, actin has been demonstrated in the subacrosomal region (Campanella et al., 1979). The same authors found actin and myosin in human sperm and suggest that myosin may possibly be present in rat sperm as well. Whether microtubules or early muscle proteins or both are responsible for the protrusions and the rhythmic motions still remains unclear. The second motile element found in spermatids, namely the axonema, could be responsible for several effects. This typical 9 + 2 axonema is obviously motile immediately after differentiation. Spermatids from Stage VIII and the following stages, integrated in the germinal epithelium, are arranged such that their flagella point to the tubular lumen and the heads to the basement membrane as mentioned above. Recently, I found that free spermatids are propelled by their flagella pushing from behind (Walt, 1980). If, in fact, the flagella of primary spermatids were to behave in the same manner in intact tubules, the flagella of the fixed spermatids would tend to displace their surroundings in the direction of the tubular lumen. Consequently, tubular fluid, sperm and probably even immotile late spermatids, still somewhat fixed to a Sertoli cell by the tubulobular complex (Russell and Clermont, 1976), could be transferred by the resultant mechanical force into the center of the lumen, where the mass of sperm is present. In addition, this spermatic flux
161 m a y be aided t o f l o w m o r e easily along t h e seminiferous tubules through the influence of m o t i l e s p e r m a t i d flagella, f o r m i n g s o m e k i n d o f a flagellated cell layer. T h u s , t h e p h a s e b e t w e e n s p e r m a t i d differe n t i a t i o n a n d s p e r m release seems t o be d o m i n a t e d n o t o n l y b y t h e Sertoli cell as t h e active m e c h a n i s m in m o v i n g of d i f f e r e n t i a t i n g g e r m cells. T h e striking m o t i o n s o f early sperm a t i d c o m p o n e n t s suggest t h a t t h e s e cells d o themselves contribute to their orientation and t r a n s f e r w i t h i n t h e germinal e p i t h e l i u m t o t h e l i b e r a t i o n a n d t r a n s p o r t o f t h e i r m a t u r e stages.
Acknowledgements T h e a u t h o r w o u l d like t o t h a n k Mrs. M. Stillhart ( D e p a r t m e n t o f N e p h r o l o g y ) , Mr. J. B e r w e r t a n d Mr. B. L o r e n z e t t i ( U n i v e r s i t y T V - T e a m ) , Miss B. Stahel, Mr. Ch. H~iberlin and Mr. H. N e f o f o u r I n s t i t u t e f o r t h e i r v a l u a b l e assistance, a n d Prof. Chr. H e d i n g e r and m y colleague Dr. A. y o n H o c h s t e t t e r f o r t h e i r critical c o m m e n t s .
References Campanella, C., G. Gabbiani, B. Baccetti, A.G. Burrini and V. Pallini: J. Submicrosc. Cytol. 11, 53-71 (1979). Ellis, L.C., J.R. Buhrley and J.L. Hargrove: Arch. Androl. 1,139--146 (1978). Fawcett, D.W., W. Anderson and D.M. Phillips: Dev. Biol. 26, 220--251 (1971). Fawcett, D.W. and D.M. Phillips: J. Reprod. Fertil. Suppl..6,405--418 (1969). Gondos, B.: Differentiation 1,177--182 (1973). Leblond~ C.P. and Y. Clermont: Ann. N.Y. Acad. Sci. 55, 548--573 (1952). McGinley, D.M., Z. Posalaky, M. Provaznik and L.D. Russell: Tissue Cell 11,741--754 (1979). Romrell, L.J. and M.H. Ross: Anat. Rec. 193, 23--42 (1979). Russell, L.D.: Anat. Rec. 194, 213--232 (1979). Russell, L.D.: Gamete Res. 3,179--202 (1980). Russell, L.D. and Y. Clermont: Anat. Rec. 185, 259-278 (1976). Setchell, B.P.: In: The Mammalian Testis. Elek Books Limited, London (1978). Setchell, B.P., T.W. Scott, J.K. Voglmayr and G.M.H. Waites: Biol. Reprod. Suppl. 1, 40--66 (1969). Voglmayr, J.K., T.W. Scott, B.P. Setchell and G.M.H. Waites: J. Reprod. Fertil. 14, 87--99 (1967). Walt, H.: Eur. J. Cell Biol. 22,559 (1980). Yochem, D.E.: Physiol. Zool. 3,309--329 (1930).