85
J. Anat. (1990), 171, pp. 85-92 With 7 figures Printed in Great Britain
Ultrastructure of the monkey vas deferens SENG-KEE LEONG AND GURMIT SINGH
Department of Anatomy, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511
(Accepted 29 December 1989) INTRODUCTION
Though the ultrastructure of the vas deferens has been extensively investigated in various species, e.g. the salamander (Zalisko & Larsen, 1988), rat (Neimi, 1965; Dym & Romrell, 1975; Kennedy, 1978; Kennedy & Heidger, 1979), rabbit (Lohiya & Mathur, 1983), dog (Murakami, Nishida, Shiromoto & Inokuchi, 1986) and monkey and/or man (Hoffer, 1970; Popovic, McLeod & Borski, 1973; Dym & Romrell, 1975; Murakami, Sugita & Hamasaki, 1982) some new features not described previously were discovered in the course of our investigation of the innervation of the monkey vas deferens. The present paper focuses on such new features. MATERIALS AND METHODS
Two male monkeys (Macaca fascicularis) weighing 3-4 kg and aged about 3 years were used in this experimental study. Small pieces of their right and left vasa deferentia were taken at the testicular and prostatic ends and immersed for one hour in a solution containing 0-1 M sodium chromate and potassium dichromate, 1 % glutaraldehyde and 0-4% paraformaldehyde adjusted to pH 7-2 and cooled to 4 'C. They were then stored overnight in 0-2 M sodium chromate/potassium dichromate buffer (pH 6) at 0-4 'C, postfixed in 2 % osmic tetroxide and processed for electron microscopy in the usual manner. The above procedure follows that of Richards & Tranzer (1975) and airns to demonstrate amine storage sites in nerve fibres which will be separately described (Leong & Singh, 1990). The sections were examined and photographed in a Philips 400T or JEOL 1200EX electron microscope. OBSERVATIONS
General features As the present observations of the ultrastructure of the monkey vas deferens are in general agreement with those of Murakami, Sugita & Hamasaki (1982), the main features of the vas deferens will be cursorily dealt with and only features not described by Murakami et al. will be elaborated. The prostatic end of the.monkey vas deferens is obviously thicker than the testicular end. At both ends, the vas deferens displays a lumen lined by an epithelium separated by a thick basal lamina from the underlying lamina propria. External to the lamina propria are three concentric layers of smooth muscle cells covered by the serosa. The mucosal lining at the testicular end is mildly folded whereas that at the prostatic end shows a very complicated folding and an accessory lumen is seen to arise from the central one. The epithelium at the testicular end is composed of pseudostratified tall columnar cells with stereocilia protruding into
86
S.-K. LEONG AND G. SINGH
Ultrastructure of monkey vas deferens 87 the lumen. Interspersed between the columnar cells are stellate basal cells. Junctional complexes between the epithelial cells are regular features. Two types of columnar cells may be distinguished in the epithelium, the principal and the mitochondria-rich cells. The principal cells may show a pale or dense cytoplasm as described in the human vas deferens (Popovic et al. 1973). The dense type of principal cell possesses prominent Golgi profiles and associated vesicles, an accumulation of mitochondria and numerous profiles of granular endoplasmic reticulum which may be arranged as stacks of compact lamellae or as grossly dilated cisterns. The apical end of such cells projects into the lumen and lies embedded among the stereocilia. Some of them, however, are far removed from the cilia, and appear to be detached from the cell and floating in the lumen. Not uncommonly, the apical end of such cells shows a bleb-like formation containing membranes arranged in a highly disorganised fashion. Such a formation has a constriction at its base and is most probably in the process of being shed from the cell body. In both the pale and dense types of columnar cells, rather dense granules of pleomorphic shape may be found. They may be situated in a supranuclear or infranuclear position. The mitochondria-rich cells resemble those seen in the human vas but some of them appear darkened. In addition to the principal and mitochondriarich cells, cells containing cellular debris in advanced stages of dissolution are also present in the epithelium. They are most likely to be intraepithelial lymphocytes which have been reported to be present in the vas deferens of the rat and monkey (Dym & Romrell, 1975) and in man (Hoffer, 1970). Sperm cells and macrophages are found in great abundance in the lumen of the prostatic end of the vas deferens. Here, spermatozoa in various stages of phagocytosis are seen in the cytoplasm of the intraluminal macrophages (Fig. 1). Such features occur most frequently in the accessory lumen arising from the central one. It appears that the accessory lumen is a reservoir wherein excess spermatozoa are phagocytosed. The columnar cells at the prostatic end possess short microvilli and contain, in the supranuclear cytoplasm, numerous membrane-bound vesicles, containing an amorphous substance of varying density. These are probably secretory granules. The cells may also contain the dense osmiophilic granules (Fig. 2) observed in the proximal end of the vas deferens. Dense lipofuscin granules, which probably represent the residual bodies of digested spermatozoa, also occur frequently in the columnar and basal cells. In the lamina propria, collagen fibres, mast cells, fibroblasts, macrophage-like cells, capillaries and scattered smooth muscle cells are commonly encountered. The mast cells may or may not be related to blood vessels. In addition to the above, there are many dense osmiophilic cells. The last-named cell type has a large nucleus surrounded by a thin peripheral rim of cytoplasm (Fig. 3). It is stellate and gives off many branching processes. In some sections, its longest process is seen to have many lateral projections (Fig. 4). The cytoplasm contains mitochondria, small dense particles, free ribosomes, granular endoplasmic reticulum (Figs. 5-7), lipofuscin granules, and Fig. 1. An intraluminal macrophage at the prostatic end of the vas deferens, containing numerous spermatozoa in various stages of digestion. Fig. 2. Dense osmiophilic granules (arrows), of pleomorphic shape, present in the infranuclear cytoplasm of a columnar cell in the epithelium at the prostatic end of the vas deferens. Fig. 3. A dense osmiophilic cell with a nerve varicosity in intimate contact with it. A layer of basal lamina indicated by long arrows is seen to surround the cell. The layer of the basal lamina separating the lamina propria from the epithelium is indicated by double arrows. Profiles of granular endoplasmic reticulum in the dense osmiophilic cell are indicated by short arrows. Another nerve varicosity (Type I) appears at the lower right hand corner of the electron micrograph. Bars for Figures 1-3: 1 ,um.
88
S.-K. LEONG AND G. SINGH
No
W .0,
PQ
Ultrastructure of monkey vas deferens
89
4-W
~~~~~~~~~~~~~~~~~~- .~ Fig. 7. Processes of dense osmiophilic cells in intimate contact with a nerve varicosity (arrow). Note the dense osmiophilic particles present in the cytoplasm of the processes. Other nerve varicosities are also present in the intercellular space on the left of the electron micrograph. Bar: 0 5 /tm.
sometimes stacks of membranes arranged in a lamellar pattern (Fig. 4). The surface membrane of the cell displays many caveolae. In many cases, a layer of basal lamina can be seen to surround it (Fig. 3) but in some cases, such a basal lamina could not be observed. The cell is often in close contact with macrophage-like cells (Fig. 5) or with the process or cell body of a smooth muscle cell and it is contacted by two types of nerve varicosities. The first type has a predominance of small agranular vesicles (measuring 29-60 nm) interspersed with some large granular vesicles (measuring 80-100 nm) which contain a dense core of variable density (Figs. 3, 5-7). These are presumably cholinergic fibres. The second type of nerve varicosity contains a Fig. 4. The processes of dense osmiophilic cells. the long process (P1) has many lateral projections. Note the stacks of membranes arranged in lamellae (indicated by arrows) in two of the processes (P1 and P2). Fig. 5. Dense osmiophilic cells in contact with macrophage-like cells (MI and M2). The arrow indicates a nerve varicosity in contact with the process of one of the dense osmiophilic cells. Fig. 6. Shows a magnified picture of the nerve varicosity indicated by the arrow in Figure 5. Bars: 1 Ism. 4
ANA 171
90
S.-K. LEONG AND G. SINGH
predominance of large, round granular vesicles (55-120 nm) and a variable number of agranular vesicles. These are probably purinergic nerves (Burnstock, 1972). Both types of nerve varicosity are in close contact with the dense osmiophilic cells. The nerve and smooth muscle membranes are often so close to each other that the interval separating them can hardly be measured. The muscle coat consists of an inner longitudinal, middle circular and an outer longitudinal layer. Close contacts between adjacent processes of the smooth muscle cells are frequently observed. There are also contacts between fibroblasts and smooth muscle cells. The electron-dense cells described above, together with mast cells, are also present in the muscle coat but to a lesser extent than occurs in the lamina propria. In many cases, the mast cells are not related to blood vessels. In fact, a mast cell may be seen to lie in close contact with a smooth muscle cell. Lastly, large numbers of nerve and collagen fibres occupy the space between muscle fibres. DISCUSSION
The epithelium The morphological features of the epithelium of the monkey vas deferens, characterised by the presence of prominent Golgi profiles, mitochondria, granular endoplasmic reticulum and secretory granules, suggest that the vas deferens may not function simply as a conduit for the transport of spermatozoa. It may play some role in the absorption of fluid from the lumen of the vas deferens, in the maturation of the spermatozoa and in spermiophagy which occurs most abundantly at the prostatic end of the vas deferens, as shown in the present study and in the study of Murakami et al. (1982). This concept has been extensively discussed by previous investigators (e.g. Hoffer, 1970; Murakami et al. 1982, 1986). The present study shows that parts of the epithelial cells may be shed off into the lumen of the vas deferens. Whether such materials would be taken up as nutrients for the spermatozoa or simply be conveyed to the outside for disposal needs to be investigated. The appearance of an abundance of mitochondria in mitochondria-rich cells indicates the high levels of energy required of them. Such energy may be necessary for fluid absorption which occurs mainly at the prostatic end of the vas deferens. It is not certain whether the darkened appearance of some of these cells indicates that they are being 'burnt out' and are in a state of degeneration. The dense pleomorphic granules noted in the principal cells at both the testicular and the prostatic ends of the vas deferens have not been described previously. They could be secretory granules but their exact nature awaits physiological elucidation. The dense cells The dense cells found in both the lamina propria and muscle coat, but more abundantly in the lamina propria, have not been described in any of the previous studies on the vas deferens. A layer of basal lamina is clearly visible around the surface membranes of many of them but appears to be absent in others. The failure to demonstrate the presence of a basal lamina around some of these cells may be due to inadequate fixation as the tissue was immersion-fixed. Though the dense cells resemble fibroblasts, having long branching processes and containing profiles of granular endoplasmic reticulum, they are not fibroblasts for no basal lamina has been seen to cover such cells. Furthermore caveolae, commonly seen on the surface of the dense cells, have not been noted in fibroblasts. Though the dense cells resemble smooth muscle cells in that both cell types display many caveolae, they cannot be smooth muscle cells of the usual type as they do not have myofilaments. The dilemma that this
91
Ultrastructure of monkey vas deferens
cell type presents is somewhat like that encountered in the interpretation of interstitial cells of Cajal present in the gastrointestinal tract (for review, see Gabella, 1981). Though the exact nature of the dense cells is unknown, it may be inferred from their rich innervation and from their branching processes that they may function as part of the contractile system of the vas deferens. Other features histamine is present at high levels in the male that demonstrated It has been the vas deferens, of different animal species including accessory sexual organs, 1969). The histamine could, in fact, be Moulton, 1965; Thomas, (Assaykeen & that mast cells are present in the monkey showed study this secreted by mast cells as to blood vessels. Also, the close contact related not are vas deferens and many of them between the mast cell and smooth muscle cells noted in this study may indicate that the mast cell could secrete histamine to act on histamine receptors present on the surface membranes of smooth muscle cells (Bhalla & Marshall, 1980; Vohra, 1981; Calvete, Hayes & Thom, 1984; Lau, Adaikan & Ratnam, 1989). Lastly, the close contacts between smooth muscle cells probably form the basis of low resistance pathways (Burnstock & Iwayama, 1971) and evidence for interfibre spread of current has been demonstrated in the mouse vas deferens (Furness & Burnstock, 1969). SUMMARY
The testicular and prostatic ends of the vasa deferentia of two male monkeys weighing 3-4 kg were examined ultrastructurally, using the method described by Richards & Tranzer (1975). The general ultrastructure of the vas deferens resembles that described previously. In addition, new features were observed. These include (i) shedding of parts of the apical portions of the epithelial cells; (ii) darkening of mitochondria-rich cells in the epithelium; (iii) presence of secretory granule-like structures in the principal cells of the epithelium; (iv) mast cells which may be associated with blood vessels or smooth muscle cells, and (v) presence of dense osmiophilic cells in the lamina propria and muscle coat, but predominantly in the former. The last-mentioned cells are stellate, having many branching processes, and are characterised by the appearance of many caveolae on their surface membranes. Basal lamina may or may not be seen to surround the cell membranes. The cells are contacted by nerve varicosities which resemble those of cholinergic and purinergic fibres. We thank the staff of the Electron Microscopic Unit of the National University of Singapore for technical assistance and Mrs M. Singh for her untiring efforts in typing the manuscript. This project was supported by a grant from the Singapore Turf Club. REFERENCES
ASSAYKEEN, T. A. & THOMAS, J. A. (1965). Endogenous histamine in male organs of reproduction. Endocrinology 76, 839-843. BHALLA, P. & MARSHALL, I. (1980). histamine H1- and H2-receptor-mediated effects in vasa deferentia from the rat, guinea-pig and rabbit. British Journal of Pharmacology 69, 304-305P. BURNSTOCK, G. & IWAYAMA, T. (1971). Fine-structural identification of autonomic nerves and their relation to smooth muscle. In Histochemistry of Nervous Transmission (ed. 0. Eranko). Progress in Brain Research 34, 389-404. CALVETE, J. A., HAYES, R. J. & THOM, S. (1984). Evidence for the occurrence of histamine Hl-receptors in human vas deferens. British Journal of Pharmacology 83 Suppl. 441P.
4.2
S.-K. LEONG AND G. SINGH 92 DYM, M. & ROMRELL, L. J. (1975). Intraepithelial lymphocytes in the male reproductive tract of rats and rhesus monkeys. Journal of Reproduction and Fertility 42, 1-7. FURNESS, J. B. & BURNSTOCK, G. (1969). A comparative study of spike potentials in response to nerve stimulation in the vas deferens of the mouse, rat and guinea-pig. Comparative Biochemistry and Physiology 31, 337-345. GABELLA, G. (1981). Structure of muscles and nerves in the gastro-intestinal tract. In Physiology of the Gastrointestinal Tract (ed. L. R. Johnson), pp. 197-241. New York: Raven Press. HOFFER, A. P. (1970). The ultrastructure of the ductus deferens in man. Biology of Reproduction 14, 425-443. KENNEDY, S. W. (1978). Regional variations in the rat vas deferens. Anatomical Record 190, 442. KENNEDY, S. W. & HEIDGER JR., P. M. (1979). Fine structural studies of the rat vas deferens. Anatomical Record 194, 159-180. LAU, L. C., ADAIKAN, P. G. & RATNAM, S. S. (1989). Effect of histamine in human vas deferens in vitro. British Journal of Urology 64, 423-427. LEONG, S. K. & SINGH, G. (1990). Innervation of the monkey vas deferens. Journal of Anatomy 171, 93-104. LOHIYA, N. K. & MATHUR, N. (1983). Excurrent duct system in male rabbit: a morphological study. Acta Europaea Fertilitatis 14, 433-441. MOULTON, B. C. (1969). Histamine levels in male reproductive organs of the rat. Endocrinology 84, 497-500. MuRAKAMI, M., NISHIDA, T., SHIROMOTO, M. & INOKUCHI, T. (1986). Scanning and transmission electron microscopic study of the ampullary region of the dog vas deferens with special reference to epithelial phagocytosis of spermatozoa and latex beads. Anatomischer Anzeiger 162, 289-296. MURAKAMI, M., SUGITA, A. & HAMASAKI, M. (1982). Scanning electron microscopic observations of the vas deferens in man and monkey with special reference to spermiophagy in its ampullary region. Scanning Electron Microscopy 111, 1333-1339. NEIMI, M. (1965). The fine structure and histochemistry of the epithelial cells of the rat vas deferens. Acta anatomica 60, 207-219. Popovic, N. A., MCLEOD, D. G. & BORSKI, A. A. (1973). Ultrastructure of the human vas deferens. Investigative Urology 10, 266-277. RicHARDs, J. G. & TRANZER, J. P. (1975). Localization of amine storage sites in the adrenergic cell body. A study of the superior cervical ganglion of the rat by fine structural cytochemistry. Journal of Ultrastructural Research 53, 204-216. VoHRA, M. M. (1981). Species differences in histamine receptors in the vas deferens. Agents and Actions 11, 208-214. ZALISKO, E. J. & LARSEN JR., J. H. (1988). Ultrastructure and histochemistry of the vas deferens of the salamander Rhyacotriton olympicus: adaptations for sperm storage. Scanning Microscopy 2, 1089-1095.
J. Anat. (1990), 171, pp. 85-92 With 7 figures Printed in Great Britain
Ultrastructure of the monkey vas deferens SENG-KEE LEONG AND GURMIT SINGH
Department of Anatomy, Faculty of Medicine, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511
(Accepted 29 December 1989) INTRODUCTION
Though the ultrastructure of the vas deferens has been extensively investigated in various species, e.g. the salamander (Zalisko & Larsen, 1988), rat (Neimi, 1965; Dym & Romrell, 1975; Kennedy, 1978; Kennedy & Heidger, 1979), rabbit (Lohiya & Mathur, 1983), dog (Murakami, Nishida, Shiromoto & Inokuchi, 1986) and monkey and/or man (Hoffer, 1970; Popovic, McLeod & Borski, 1973; Dym & Romrell, 1975; Murakami, Sugita & Hamasaki, 1982) some new features not described previously were discovered in the course of our investigation of the innervation of the monkey vas deferens. The present paper focuses on such new features. MATERIALS AND METHODS
Two male monkeys (Macaca fascicularis) weighing 3-4 kg and aged about 3 years were used in this experimental study. Small pieces of their right and left vasa deferentia were taken at the testicular and prostatic ends and immersed for one hour in a solution containing 0-1 M sodium chromate and potassium dichromate, 1 % glutaraldehyde and 0-4% paraformaldehyde adjusted to pH 7-2 and cooled to 4 'C. They were then stored overnight in 0-2 M sodium chromate/potassium dichromate buffer (pH 6) at 0-4 'C, postfixed in 2 % osmic tetroxide and processed for electron microscopy in the usual manner. The above procedure follows that of Richards & Tranzer (1975) and airns to demonstrate amine storage sites in nerve fibres which will be separately described (Leong & Singh, 1990). The sections were examined and photographed in a Philips 400T or JEOL 1200EX electron microscope. OBSERVATIONS
General features As the present observations of the ultrastructure of the monkey vas deferens are in general agreement with those of Murakami, Sugita & Hamasaki (1982), the main features of the vas deferens will be cursorily dealt with and only features not described by Murakami et al. will be elaborated. The prostatic end of the.monkey vas deferens is obviously thicker than the testicular end. At both ends, the vas deferens displays a lumen lined by an epithelium separated by a thick basal lamina from the underlying lamina propria. External to the lamina propria are three concentric layers of smooth muscle cells covered by the serosa. The mucosal lining at the testicular end is mildly folded whereas that at the prostatic end shows a very complicated folding and an accessory lumen is seen to arise from the central one. The epithelium at the testicular end is composed of pseudostratified tall columnar cells with stereocilia protruding into
86
S.-K. LEONG AND G. SINGH
Ultrastructure of monkey vas deferens 87 the lumen. Interspersed between the columnar cells are stellate basal cells. Junctional complexes between the epithelial cells are regular features. Two types of columnar cells may be distinguished in the epithelium, the principal and the mitochondria-rich cells. The principal cells may show a pale or dense cytoplasm as described in the human vas deferens (Popovic et al. 1973). The dense type of principal cell possesses prominent Golgi profiles and associated vesicles, an accumulation of mitochondria and numerous profiles of granular endoplasmic reticulum which may be arranged as stacks of compact lamellae or as grossly dilated cisterns. The apical end of such cells projects into the lumen and lies embedded among the stereocilia. Some of them, however, are far removed from the cilia, and appear to be detached from the cell and floating in the lumen. Not uncommonly, the apical end of such cells shows a bleb-like formation containing membranes arranged in a highly disorganised fashion. Such a formation has a constriction at its base and is most probably in the process of being shed from the cell body. In both the pale and dense types of columnar cells, rather dense granules of pleomorphic shape may be found. They may be situated in a supranuclear or infranuclear position. The mitochondria-rich cells resemble those seen in the human vas but some of them appear darkened. In addition to the principal and mitochondriarich cells, cells containing cellular debris in advanced stages of dissolution are also present in the epithelium. They are most likely to be intraepithelial lymphocytes which have been reported to be present in the vas deferens of the rat and monkey (Dym & Romrell, 1975) and in man (Hoffer, 1970). Sperm cells and macrophages are found in great abundance in the lumen of the prostatic end of the vas deferens. Here, spermatozoa in various stages of phagocytosis are seen in the cytoplasm of the intraluminal macrophages (Fig. 1). Such features occur most frequently in the accessory lumen arising from the central one. It appears that the accessory lumen is a reservoir wherein excess spermatozoa are phagocytosed. The columnar cells at the prostatic end possess short microvilli and contain, in the supranuclear cytoplasm, numerous membrane-bound vesicles, containing an amorphous substance of varying density. These are probably secretory granules. The cells may also contain the dense osmiophilic granules (Fig. 2) observed in the proximal end of the vas deferens. Dense lipofuscin granules, which probably represent the residual bodies of digested spermatozoa, also occur frequently in the columnar and basal cells. In the lamina propria, collagen fibres, mast cells, fibroblasts, macrophage-like cells, capillaries and scattered smooth muscle cells are commonly encountered. The mast cells may or may not be related to blood vessels. In addition to the above, there are many dense osmiophilic cells. The last-named cell type has a large nucleus surrounded by a thin peripheral rim of cytoplasm (Fig. 3). It is stellate and gives off many branching processes. In some sections, its longest process is seen to have many lateral projections (Fig. 4). The cytoplasm contains mitochondria, small dense particles, free ribosomes, granular endoplasmic reticulum (Figs. 5-7), lipofuscin granules, and Fig. 1. An intraluminal macrophage at the prostatic end of the vas deferens, containing numerous spermatozoa in various stages of digestion. Fig. 2. Dense osmiophilic granules (arrows), of pleomorphic shape, present in the infranuclear cytoplasm of a columnar cell in the epithelium at the prostatic end of the vas deferens. Fig. 3. A dense osmiophilic cell with a nerve varicosity in intimate contact with it. A layer of basal lamina indicated by long arrows is seen to surround the cell. The layer of the basal lamina separating the lamina propria from the epithelium is indicated by double arrows. Profiles of granular endoplasmic reticulum in the dense osmiophilic cell are indicated by short arrows. Another nerve varicosity (Type I) appears at the lower right hand corner of the electron micrograph. Bars for Figures 1-3: 1 ,um.
88
S.-K. LEONG AND G. SINGH
No
W .0,
PQ
Ultrastructure of monkey vas deferens
89
4-W
~~~~~~~~~~~~~~~~~~- .~ Fig. 7. Processes of dense osmiophilic cells in intimate contact with a nerve varicosity (arrow). Note the dense osmiophilic particles present in the cytoplasm of the processes. Other nerve varicosities are also present in the intercellular space on the left of the electron micrograph. Bar: 0 5 /tm.
sometimes stacks of membranes arranged in a lamellar pattern (Fig. 4). The surface membrane of the cell displays many caveolae. In many cases, a layer of basal lamina can be seen to surround it (Fig. 3) but in some cases, such a basal lamina could not be observed. The cell is often in close contact with macrophage-like cells (Fig. 5) or with the process or cell body of a smooth muscle cell and it is contacted by two types of nerve varicosities. The first type has a predominance of small agranular vesicles (measuring 29-60 nm) interspersed with some large granular vesicles (measuring 80-100 nm) which contain a dense core of variable density (Figs. 3, 5-7). These are presumably cholinergic fibres. The second type of nerve varicosity contains a Fig. 4. The processes of dense osmiophilic cells. the long process (P1) has many lateral projections. Note the stacks of membranes arranged in lamellae (indicated by arrows) in two of the processes (P1 and P2). Fig. 5. Dense osmiophilic cells in contact with macrophage-like cells (MI and M2). The arrow indicates a nerve varicosity in contact with the process of one of the dense osmiophilic cells. Fig. 6. Shows a magnified picture of the nerve varicosity indicated by the arrow in Figure 5. Bars: 1 Ism. 4
ANA 171
90
S.-K. LEONG AND G. SINGH
predominance of large, round granular vesicles (55-120 nm) and a variable number of agranular vesicles. These are probably purinergic nerves (Burnstock, 1972). Both types of nerve varicosity are in close contact with the dense osmiophilic cells. The nerve and smooth muscle membranes are often so close to each other that the interval separating them can hardly be measured. The muscle coat consists of an inner longitudinal, middle circular and an outer longitudinal layer. Close contacts between adjacent processes of the smooth muscle cells are frequently observed. There are also contacts between fibroblasts and smooth muscle cells. The electron-dense cells described above, together with mast cells, are also present in the muscle coat but to a lesser extent than occurs in the lamina propria. In many cases, the mast cells are not related to blood vessels. In fact, a mast cell may be seen to lie in close contact with a smooth muscle cell. Lastly, large numbers of nerve and collagen fibres occupy the space between muscle fibres. DISCUSSION
The epithelium The morphological features of the epithelium of the monkey vas deferens, characterised by the presence of prominent Golgi profiles, mitochondria, granular endoplasmic reticulum and secretory granules, suggest that the vas deferens may not function simply as a conduit for the transport of spermatozoa. It may play some role in the absorption of fluid from the lumen of the vas deferens, in the maturation of the spermatozoa and in spermiophagy which occurs most abundantly at the prostatic end of the vas deferens, as shown in the present study and in the study of Murakami et al. (1982). This concept has been extensively discussed by previous investigators (e.g. Hoffer, 1970; Murakami et al. 1982, 1986). The present study shows that parts of the epithelial cells may be shed off into the lumen of the vas deferens. Whether such materials would be taken up as nutrients for the spermatozoa or simply be conveyed to the outside for disposal needs to be investigated. The appearance of an abundance of mitochondria in mitochondria-rich cells indicates the high levels of energy required of them. Such energy may be necessary for fluid absorption which occurs mainly at the prostatic end of the vas deferens. It is not certain whether the darkened appearance of some of these cells indicates that they are being 'burnt out' and are in a state of degeneration. The dense pleomorphic granules noted in the principal cells at both the testicular and the prostatic ends of the vas deferens have not been described previously. They could be secretory granules but their exact nature awaits physiological elucidation. The dense cells The dense cells found in both the lamina propria and muscle coat, but more abundantly in the lamina propria, have not been described in any of the previous studies on the vas deferens. A layer of basal lamina is clearly visible around the surface membranes of many of them but appears to be absent in others. The failure to demonstrate the presence of a basal lamina around some of these cells may be due to inadequate fixation as the tissue was immersion-fixed. Though the dense cells resemble fibroblasts, having long branching processes and containing profiles of granular endoplasmic reticulum, they are not fibroblasts for no basal lamina has been seen to cover such cells. Furthermore caveolae, commonly seen on the surface of the dense cells, have not been noted in fibroblasts. Though the dense cells resemble smooth muscle cells in that both cell types display many caveolae, they cannot be smooth muscle cells of the usual type as they do not have myofilaments. The dilemma that this
91
Ultrastructure of monkey vas deferens
cell type presents is somewhat like that encountered in the interpretation of interstitial cells of Cajal present in the gastrointestinal tract (for review, see Gabella, 1981). Though the exact nature of the dense cells is unknown, it may be inferred from their rich innervation and from their branching processes that they may function as part of the contractile system of the vas deferens. Other features histamine is present at high levels in the male that demonstrated It has been the vas deferens, of different animal species including accessory sexual organs, 1969). The histamine could, in fact, be Moulton, 1965; Thomas, (Assaykeen & that mast cells are present in the monkey showed study this secreted by mast cells as to blood vessels. Also, the close contact related not are vas deferens and many of them between the mast cell and smooth muscle cells noted in this study may indicate that the mast cell could secrete histamine to act on histamine receptors present on the surface membranes of smooth muscle cells (Bhalla & Marshall, 1980; Vohra, 1981; Calvete, Hayes & Thom, 1984; Lau, Adaikan & Ratnam, 1989). Lastly, the close contacts between smooth muscle cells probably form the basis of low resistance pathways (Burnstock & Iwayama, 1971) and evidence for interfibre spread of current has been demonstrated in the mouse vas deferens (Furness & Burnstock, 1969). SUMMARY
The testicular and prostatic ends of the vasa deferentia of two male monkeys weighing 3-4 kg were examined ultrastructurally, using the method described by Richards & Tranzer (1975). The general ultrastructure of the vas deferens resembles that described previously. In addition, new features were observed. These include (i) shedding of parts of the apical portions of the epithelial cells; (ii) darkening of mitochondria-rich cells in the epithelium; (iii) presence of secretory granule-like structures in the principal cells of the epithelium; (iv) mast cells which may be associated with blood vessels or smooth muscle cells, and (v) presence of dense osmiophilic cells in the lamina propria and muscle coat, but predominantly in the former. The last-mentioned cells are stellate, having many branching processes, and are characterised by the appearance of many caveolae on their surface membranes. Basal lamina may or may not be seen to surround the cell membranes. The cells are contacted by nerve varicosities which resemble those of cholinergic and purinergic fibres. We thank the staff of the Electron Microscopic Unit of the National University of Singapore for technical assistance and Mrs M. Singh for her untiring efforts in typing the manuscript. This project was supported by a grant from the Singapore Turf Club. REFERENCES
ASSAYKEEN, T. A. & THOMAS, J. A. (1965). Endogenous histamine in male organs of reproduction. Endocrinology 76, 839-843. BHALLA, P. & MARSHALL, I. (1980). histamine H1- and H2-receptor-mediated effects in vasa deferentia from the rat, guinea-pig and rabbit. British Journal of Pharmacology 69, 304-305P. BURNSTOCK, G. & IWAYAMA, T. (1971). Fine-structural identification of autonomic nerves and their relation to smooth muscle. In Histochemistry of Nervous Transmission (ed. 0. Eranko). Progress in Brain Research 34, 389-404. CALVETE, J. A., HAYES, R. J. & THOM, S. (1984). Evidence for the occurrence of histamine Hl-receptors in human vas deferens. British Journal of Pharmacology 83 Suppl. 441P.
4.2
S.-K. LEONG AND G. SINGH 92 DYM, M. & ROMRELL, L. J. (1975). Intraepithelial lymphocytes in the male reproductive tract of rats and rhesus monkeys. Journal of Reproduction and Fertility 42, 1-7. FURNESS, J. B. & BURNSTOCK, G. (1969). A comparative study of spike potentials in response to nerve stimulation in the vas deferens of the mouse, rat and guinea-pig. Comparative Biochemistry and Physiology 31, 337-345. GABELLA, G. (1981). Structure of muscles and nerves in the gastro-intestinal tract. In Physiology of the Gastrointestinal Tract (ed. L. R. Johnson), pp. 197-241. New York: Raven Press. HOFFER, A. P. (1970). The ultrastructure of the ductus deferens in man. Biology of Reproduction 14, 425-443. KENNEDY, S. W. (1978). Regional variations in the rat vas deferens. Anatomical Record 190, 442. KENNEDY, S. W. & HEIDGER JR., P. M. (1979). Fine structural studies of the rat vas deferens. Anatomical Record 194, 159-180. LAU, L. C., ADAIKAN, P. G. & RATNAM, S. S. (1989). Effect of histamine in human vas deferens in vitro. British Journal of Urology 64, 423-427. LEONG, S. K. & SINGH, G. (1990). Innervation of the monkey vas deferens. Journal of Anatomy 171, 93-104. LOHIYA, N. K. & MATHUR, N. (1983). Excurrent duct system in male rabbit: a morphological study. Acta Europaea Fertilitatis 14, 433-441. MOULTON, B. C. (1969). Histamine levels in male reproductive organs of the rat. Endocrinology 84, 497-500. MuRAKAMI, M., NISHIDA, T., SHIROMOTO, M. & INOKUCHI, T. (1986). Scanning and transmission electron microscopic study of the ampullary region of the dog vas deferens with special reference to epithelial phagocytosis of spermatozoa and latex beads. Anatomischer Anzeiger 162, 289-296. MURAKAMI, M., SUGITA, A. & HAMASAKI, M. (1982). Scanning electron microscopic observations of the vas deferens in man and monkey with special reference to spermiophagy in its ampullary region. Scanning Electron Microscopy 111, 1333-1339. NEIMI, M. (1965). The fine structure and histochemistry of the epithelial cells of the rat vas deferens. Acta anatomica 60, 207-219. Popovic, N. A., MCLEOD, D. G. & BORSKI, A. A. (1973). Ultrastructure of the human vas deferens. Investigative Urology 10, 266-277. RicHARDs, J. G. & TRANZER, J. P. (1975). Localization of amine storage sites in the adrenergic cell body. A study of the superior cervical ganglion of the rat by fine structural cytochemistry. Journal of Ultrastructural Research 53, 204-216. VoHRA, M. M. (1981). Species differences in histamine receptors in the vas deferens. Agents and Actions 11, 208-214. ZALISKO, E. J. & LARSEN JR., J. H. (1988). Ultrastructure and histochemistry of the vas deferens of the salamander Rhyacotriton olympicus: adaptations for sperm storage. Scanning Microscopy 2, 1089-1095.
