TISSUE & CELL 1977 9 (3) 475-498 Published by Longman Group Ltd. Printed in Great Britain

LONNIE

RUSSELL

OBSERVATIONS ON RAT SERTOLI ECTOPLASMIC (‘JUNCTIONAL’) SPECIALIZATIONS IN THEIR ASSOCIATION WITH GERM CELLS OF THE RAT TESTIS ABSTRACT. The ectoplasmic (‘junctional’) specialization, a subsurface modification of the Sertoli cell that is often seen facing germ cells, was studied in relation to the development and maturation of these germ cells. This structure is composed of subsurface bundles of filaments and more deeply placed endoplasmic reticulum. The data indicate that these subsurface modifications of Sertoli cells are reutilized in a cyclic fashion, being transferred from their position facing late spermatids to one opposing less mature germ cells. Ectoplasmic specializations appeared to function mechanically in grasping the heads of the spermatids which are undergoing the elongation and maturation phases of spermiogenesis rather than in actually attaching Sertoli cells to these germ cells. It is postulated that the ectoplasmic specialization imparts rigidity to that area of the Sertoli cell that surrounds the head region of the germ cell, forming a recess and a mantle by which the germ cell may be moved toward the base or toward the surface of the seminiferous epithelium. The observed linkage of microtubules to the cisternae of the complex provided a morphological basis for the changes in the cytoarchitecture of the Sertoli cell, which must accompany these movements.

Introduction Sertoli cells, in their relationship to other Sertoli cells and to some germ cells, form a characteristic specialization just deep to the plasma membrane. The earliest descriptions of this structure as seen at the interface of two adjoining Sertoli cells were provided by Brokelmann (1961, 1963) and Nicander (1963). A few years later it was given the name ‘junctional specialization’ by Flickinger and Fawcett (1967). This term, or the term Sertoli junction, is widely used to describe this cell modification (Fawcett et al., 1970; Ross, 1970, 1976; Ross and Dobler, 1975; Toyama, 1976; Nicander, 1967) and has also come into usage to describe a specialization of similar appearance on the surface aspect of the Sertoli cells when related to germ cells (Burgos et al., 1970; Department of Anatomy, University of Miami, School of Medicine, Miami, Florida 33152. Send offprint request to: Department of Physiology, Southern Illinois University at Carbondale, Carbondale, III. 62901. Received 9 February 1977. 475

Fawcett, 1970; Flickinger, 1967; Ross and Dobler, 1975; Ross, 1976, 1977; Russell, 1977a, 1977b). In this report, this structure will be referred to as an ectoplasmic specialization or in some cases simply specialization, whether seen facing another Sertoli cell or a germ cell. The most thorough description of ectoplasmic specializations was provided by Dym and Fawcett (1970) when describing the relationship of adjacent Sertoli cells near the base of the seminiferous tubule. Subjacent to the plasma membranes of each cell, were regularly spaced bundles of hexagonally packed filaments. Deep to the filamentous layer, and parallel with the plasma membrane, was a saccule of endoplasmic reticulum which contained ribosomes on its deep aspect and was spaced about 500 A from the Sertoli plasma membrane. At the level of ectoplasmic specializations between Sertoli cells, ‘narrow junctions’ (90 A intercellular space), gap junctions and focal tight junctions were described. The ectoplasmic specializations and associated

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junctions were termed a ‘junctional complex’ by Dym and Fawcett. These authors also showed that the tight junctions referred to above were effective in preventing tracer molecules of high molecular weight from entering the tubular lumen; thus, the junctions were thought to be the structural basis for the ‘blood-testis’ barrier. The ectoplasmic specializations of Sertoli cells, whether facing other Sertoli cells or germ cells, are almost identical in appearance. It should be pointed out that junctions (of the classical sense, Farquhar and Palade, 1963) have never been described between germ cells and Sertoli cells at sites where ectoplasmic specializations face germ cells. The role of the ectoplasmic specializations is unclear. Brokelmann (1961) felt that these structures, seen facing the heads of maturing spermatids, may play some role in sperm release. Most investigators believe that the ectoplasmic specializations, in some way as yet undefined, are attachment devices between germ cells and Sertoli cells (Nicander, 1967; Sapsford and Rae, 1969; Fawcett and Phillips, 1969; Dym and Fawcett, 1970; Ross, 1970, 1976, 1977; Ross and Dobler, 1975). Ross and Dobler (I 975) have postulated that junctional specializations first become related to germ cells as the latter (spermatocytes) cross the blood-testis barrier during their transit from the basal to the adluminal compartment of the testis. Once this relationship was established, the germ cells would remain attached throughout the maturation process until they were released into the tubular lumen. Russell has questioned the validity of this theory on the basis that Sertoli ectoplasmic specializations were not seen facing spermatocytes as the latter moved from the basal to the adluminal compartment (1977b). Instead, numerous attachment devices of the desmosome type were found to bind Sertoli cells to the spermatocytes at this time (Russell, 1977a). Toyama (1976) has recently shown that the filaments of the ectoplasmic specialization bind heavy meromyosin, indicating that these filaments are actin-like and most likely contractile in function. He postulated that these filaments may act as a gripping force on the spermatid, thus holding it in place until spermiation. Gravis et al. (1976) provided some support for this theory when they demonstrated (by cytochemical

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means) the presence of ATPases in association with filaments of the ectoplasmic specialization. They speculated that these enzymes might be important in providing energy for filament motility. The present study provides new observations on ectoplasmic specializations during the cycle of the seminiferous epithelium (as described by Leblond and Clermont, 1952). Data are presented which indicate that these structures are recycled during the spermatogenie process, and that they function to maintain recesses for elongated germ cells. In spite of an extensive search, there was no evidence to indicate that ectoplasmic specializations are junctions which act to bind cells together, but it was felt that they might provide a mechanical gripping force on the spermatid head. Materials and Methods Tissue preparation methods

using

stutdarcl

fixufiott

Normal adult (300-400 g) SpragueeDawley rats were utilized in this study. Five animals were anesthetized with sodium pentobarbital and the testes fixed by vascular perfusion through the abdominal aorta by a retrograde method (Vitale et al., 1973). First the testes were cleared with 0.9 M saline solution followed by fixation with 5”,, glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.4). Testes were removed, cut into small pieces with a razor blade and fixed in the perfusate for an additional l-2 hr at room temperature. The tissue was washed in three changes of buffer overnight in most cases, and post-fixed in 2”‘,, 0~04. Subsequent dehydration was performed in ascending concentrations of ethanol and propylene oxide. The tissue was infiltrated with a mixture of propylene oxide and Epon and embedded in Epon. One micron thick sections were stained with toluidine blue and viewed with a light microscope. Individual tubules containing cells at known stages of the cycle (classification of Leblond and Clermont, 1952) were selected for examination under the electron microscope. Silvergold, and in some cases silver-grey, appearing thin sections (cut on a Porter-Blum MT-2 ultra-microtome) were stained with uranyl acetate and lead citrate, and examined with a Hitachi-l 1C electron microscope.

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Tissue preparations using hypertonic solutions

Fresh testes from anesthetized adult Sprague Dawley rats were removed from the abdominal cavity and dissected free of the covering tunic. A small portion of each testis was placed on dental wax and finely minced with a razor blade. The resulting jelly-like mass was washed in a vial containing 0.2 M sodium cacodylate buffer (pH 7.4) with 10% sucrose added; after five minutes in this solution, the tissue was transferred to 5 % glutaraldehyde (with 10% sucrose added) for thirty minutes. The osmication, dehydration and embedding steps which followed were the same as those described above. Observations Initial appearance of Sertoli ectoplasmic specializations (facing germ cells) during the cycle of the seminiferous epithelium

In previous studies, this author (Russell, 1977b, 1977c) has examined the spermatocyte population of cells in the rat testis. Although not directly related to the topic under consideration (and thus not reported), it was found that ectoplasmic specializations of Sertoli cells faced late pachytene spermatocytes, yet were not related to many other classes of prophase spermatocytes (leptotene, zygotene, etc.). Since it was thought important to determine precisely when in the spermatogenic process that Sertoli ectoplasmic specializations could first be seen facing germ cells, a careful study was undertaken to make more specific observations. Ectoplasmic specializations were never observed facing preleptotene, leptotene, zygotene, and early pachytene spermatocytes. As a general rule, however, most late pachytene spermatocytes demonstrated some surface area in apposition to ectoplasmic specializations (Figs. 1 and 3). Mid-pachytene spermatocytes and neighboring Sertoli cells were examined to determine the precise stage of the cycle in which ectoplasmic specializations first became aligned in apposition to spermatocytes. It was observed that Sertoli ectoplasmic specializations made their first appearance facing spermatocytes in Stage VII (pachytenes), but only late in the stage. They were always seen facing spermatocytes which were more advanced in development than the Stage VII pachytene

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spermatocytes, although with the thin section techniques utilized, it was not possible to determine if a Sertoli cell modification of this type was in apposition to every spermatocyte. Characterization of Sertoli ectoplasmic specializations (opposing germ cells)

Although essentially similar in appearance to the Sertoli-Sertoli ectoplasmic specializations, those specializations seen in relation to germ cells displayed considerably fewer ribosomes on the membranes of the endoplasmic reticulum. Only an occasional ribosome or small grouping of ribosomes were seen along the deep aspect of the cisternae (Figs. 2, 5). Micrographs taken at an original magnification of over x 30,000 and enlarged from 3-5 times, were used to measure the diameter of the hexagonally packed filaments of the specialization. These filaments were found to be between 70-90 8, across. Several longitudinal profiles of filaments were observed which appeared to be inserted into either the saccule of endoplasmic reticulum or the Sertoli plasma membrane (Figs. 4, 5). Junctions of the classical type (Farquhar and Palade, 1963) were not seen between two cells at sites of Sertoli ectoplasmic specializations (Figs. l-5, 8-10). The intercellular space was carefully examined in sections cut perpendicular to the plasma membranes. Features which would normally suggest some type of binding substance between the two cells were found to be missing. The space appeared electron translucent, lacking any apparent ‘connecting material’ which might be associated with an adhering type junction. Fuzzy material, like that comprising the cell coat (glycocalyx) of plasma membranes, was the only external feature observed that extended out from the surfaces of the two cells. The width of the intercellular space was quite variable in the relationship of spermatocytes and early spermatids with Sertoli cells (at ectoplasmic sites), generally measuring from 150-300 A across. In some regions the width of the space approached 1 pm, although it was not known if this spacing was artifactual (Fig. 3). In its relationship with the heads of the more mature, elongating spermatids, the intercellular space was narrower (10&l 50 A) and characteristic-

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ally more uniform (Figs. 2, 4, 5, 9). Elsewhere in regions of elongating spermatids, where the facing Sertoli specialization was not present, the width of the intercellular space was quite variable (Figs. 8, 9) and more closely resembled that described above between spermatocytes, non-elongate spermatids, and Sertoli cells at ectoplasmic

sites. In many cases, microtubules were observed just deep to (lOCk500 A away from) the ectoplasmic specializations (Figs. 2, 10). Sertoli ectoplasmic specialization during the cycle of the seminiferous epithelium

As stated above, Sertoli ectoplasmic specializations were first observed in relation to

Figs. I through 23. Electron micrographs showing the relationship of Sertoli cells to germ cells at various stages of the cycle (classification of Leblond and Clermont, 1952). Fig. 1.An ectoplasmic specialization (arrowheads) is seen at the surface of the Sertoli cell, facing a Stage XI pachytene spermatocyte. x 7000. Fig. 2. A Sertoli ectoplasmic specialization is seen facing the acrosomal region (a) of a late (step 18) spermatid. The filamentous component (asterisks) and elongated cisterna of endoplasmic reticulum (er) (containing scattered ribosomes (r) on its surface) are easily identified in cross-section. Numerous microtubules (mt) are found deep to the cisterna of endoplasmic reticulum. The intercellular space (arrowheads) between the two cells is uniform in width, and appears electron translucent when the section is perpendicular to the plane of the membranes. x 70,000. Fig. 3. A Sertoli (S) ectoplasmic specialization (arrowheads) is seen facing a pachytene spermatocyte in Stage VIII. The width of the intercellular space (is) between the two cells is highly variable and the two plasma membranes describe an irregular course. A small desmosome-like contact is indicated by the arrow. x 15,500. Figs. 4 and 5. Features indicated in this micrograph (showing the relationship of a step 9 spermatid to Sertoli ectoplasmic specialization) include cisternae of endoplasmic reticulum (er), ribosomes (r), filaments (f), Sertoli plasma membrane (large arrow), intercellular space (asterisk), spermatid plasma membrane (small arrow), acrosomal cap (ac), spermatid nucleus (II). Sites of filament attachment are indicated by the arrowheads. x 90,000 and x 66,000 respectively. Figs. 6 and 7. Survey micrographs showing representative areas of Stage V and Stage IX tubules respectively. Step 5 (early generation) and step 17 (late generation) are seen in Fig. 6, and step 9 spermatids in Fig. 7. Sertoli ectoplasmic specializations (arrowheads) face the acrosomal system of all step 9 and step 17 spermatids but are seen only infrequently in apposition to step 5 spermatids. Sheet-like processes (p) of Sertoli cytoplasm are seen around most areas of step 5 spermatids, whereas much larger Sertoli recesses are related to the head regions of step 9 and especially step 17 spermatids. Each x 4000. Figs. 8 and 9. In these Stage VIII (Fig. 8) and Stage IX (Fig. 9) tubules, the ectoplasmic specializations (arrowheads) are seen overlying the acrosomal system (a) with little specialization facing non-acrosomal regions. In Fig. 8, shallow recesses within the body of the Sertoli cell partially envelope the head of the elongating spermatid. Small processes (p) of Sertoli cytoplasm are related to the trailing cytoplasmic mass of the spermatid. In Fig. 9, the intercellular space at sites of ectoplasmic specializations (small arrows) is uniform and generally narrower than at sites not showing the opposing specialization (large arrows). A desmosome-like (d) structure is seen at a non-ectoplasmic specialization site. x 6400 and x 14,000 respectively. Fig. 10. The head of a longitudinally sectioned step 12 spermatid is embedded within a Sertoli recess, whereas the trailing cytoplasmic mass is related to much smaller Sertoli processes (p). The extensive surface contact between Sertoli ectoplasmic specializations and the spermatid is indicated by arrowheads. The extent of the acrosomal cap is indicated by white dots. Ectoplasmic specializations (arrows) are also seen facing a pachytene spermatocyte as well as a cross-sectional profile of a step 12 spermatid. x 18,000.

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pachytene spermatocytes in late Stage VII. They were also seen facing more mature pachytene spermatocytes (Figs. 1, 3), diplotene spermatocytes, secondary spermatocytes, meiotic figures, and all spermatids up to step 19 of spermiogenesis (Figs. 4-11). Ross (1976) has documented the presence of these structures (in the mouse) in relation to the spermatid population of cells. In the present study it was noted that fewer secondary spermatocytes (in sections) showed areas facing ectoplasmic specialization than did pachytene spermatocytes. Likewise, even fewer (in contrast to secondary spermatocytes) step 1 spermatids (of Stage I) appeared in contact with ectoplasmic specializations; in fact, by this stage these structures were only rarely observed facing young spermatids (Fig. 6). When present, they were generally only related to the early spermatids over a small area of the surface of the cell. It was not until examining Stage VIII tubules (and tubules more advanced), that it was found that the chance of sectioning through Sertoli ectoplasmic specialization had substantially increased (Figs. 7-10). At this particular time in spermiogenesis, the nucleus of the step 8 spermatid became acentric within the cell. The acrosomal system approximated the spermatid plasma membrane, and this region of the cell became oriented toward the limiting membrane of the tubule. Shortly thereafter, profiles were observed in which the acrosoma1 system was seen facing Sertoli ectoplasmic specializations. Thus, by mid and late Stage VIII and by Stage IX, any section through the acrosome also transversed the Sertoli ectoplasmic specializations. The extensive nature of this relationship in elongating spermatids is demonstrated in Figs. 7 and 8 and should be compared with that seen in non-elongate spermatids as shown in Fig. 6. In Stages I through VIII, the non-elongate spermatids were related to thin lateral processes of the Sertoli cells (Fig. 6) and bound to these processes by desmosome-like contacts (Russell, 1977a). In Stage IX, the heads of the spermatids became lodged within recesses of the bodies of the Sertoli cells, whereas the trailing cytoplasmic masses of the spermatids were related to smaller apical and lateral Sertoli processes (Figs. 7,8). Desmosome-like contacts, seen between

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Sertoli cells and spermatids in earlier Stages (I-VIII), appeared rudimentary and were only rarely noted between the two cell types in Stage IX (Fig. 9). They were almost never observed in subsequent stages. As the elongation and condensation phase of spermiogenesis continued in Stages X through XIV, the surface area of the spermatid nucleus (head) increased substantially (Fig. 10). The acrosomal system spread out over the nucleus to cover most of its external surface. A close approximation of the acrosome to the spermatid plasma membrane was maintained as the area of contact of the acrosome with the spermatid plasma membrane increased. The opposing Sertoli ectoplasmic specialization became more extensive and covered the expanded acrosomal system (Fig. 10). At these stages, the Sertoli recesses (containing the spermatid heads) were easily visualized, where it was obvious that the heads of the spermatids were deeply embedded in the ‘trunk’ of the Sertoli cells. The trailing cytoplasmic masses of the spermatids were always related to the much smaller apical and lateral processes of the Sertoli cells (Fig. 10). The head regions of elongating spermatids (in steps 10-13) were embedded deeply within the Sertoli cells, and were situated at a level within the seminiferous epithelium that was between pachytene spermatocytes. Following the meiotic divisions of the latter cells, the heads of the late spermatids were seen to be positioned among step 1 spermatids, where they appeared quite superficial in the seminiferous epithelium. Examination of this area with the electron microscope showed that the heads of these cells were overlain by Sertoli ectoplasmic specialization, and were still deeply embedded within Sertoli recesses. In Stage II the spermatids were moved (or moved) toward the base of the tubule to assume a position at the level of the new generation of pachytene spermatocytes. It was not until the end of Stage VI of the cycle that the heads of the late spermatids (step 19) were seen to change position again. At this time they were moved (or moved) a considerable distance, reaching the surface of the seminiferous epithelium. These aforementioned positional changes of late spermatids have been previously described by Leblond and Clermont (1952). In Stage VII, the spermatids were related

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to apical expansions of Sertoli cytoplasm which were connected to the main body of the Sertoli cell by a narrow stalk. Even in this position at the surface of the seminiferous epithelium, the spermatid heads were embedded in Sertoli recesses (see Russell and Clermont, 1976). During Stage VII and Stage VIII, the relationship between the late spermatids and the Sertoli cells changed. In late Stage VII, there was a shift in the position of the Sertoli ectoplasmic specializations (Fawcett and Phillips, 1969; Ross, 1976; Russell and Clermont, 1976). Having lost their position facing the heads of the elongated spermatids, the ectoplasmic specializations (by late Stage VII) were seen in various positions along the surfaces of the apical Sertoli expansions and apical Sertoli stalks. By the completion of Stage VII, the ectoplasmic specializations had gained a position considerably closer to the base of the tubule. Here, the specializations faced

residual cytoplasmic masses or the free surfaces of the apical Sertoli stalks (Fig. 11). In some cases they had lost their position on the surface aspects of the Sertoli cells and were internalized (Figs. 11, 12). Several profiles were observed in which the specializations were in partial apposition to the early generation of spermatids (steps 7 and 9; Fig. 12) or pachytene spermatocytes (of Stage VII). Concomitant with the shift of the ectoplasmic specializations away from the heads of the spermatids, the recesses in which the spermatid heads were embedded (within the apical droplets) were erased (Fig. 13), and shortly thereafter, sperm were released (Fawcett and Phillips, 1969; Ross, 1976; Russell and Clermont, 1976). Experiments

Examination

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Fig. 1 I. Late Stage VII tubule showing an area at the surface of the seminiferous epithelium exhibiting the early (step 7) and late (step 19) generations of spermatids. The heads of the step 19 spermatids are embedded in an apical Sertoli expansion (Se). The ectoplasmic specializations (arrowheads) are seen at the free surfaces of the Sertoli stalk, facing the spermatid residual cytoplasm (rc), internalized within the cell process (arrows), and in apposition to the late step 7 spermatids, but not in a position facing the heads of the late spermatids. x 4200. Fig. 12. A Stage VII tubule showing a Sertoli ectoplasmic specialization facing two late step 7 spermatids. Between the two cells is an area in which the specialization is seen at the free surface of the Sertoli cell, facing the intercellular space (is). Another specialization is internalized (arrow). x 9000. Fig. 13. Surface of the seminiferous epithelium in Stage VIII showing the late (step 19) spermatids, residual cytoplasm (rc) and apical Sertoli processes (p). The recesses in which the spermatid heads were embedded (see Fig. 1 I) are no longer evident. x 4900. Fig. 14. Desmosome-like (arrowheads) contact between a Sertoli cell and a late pachytene spermatocyte after treatment of tissue with a hypertonic solution. The intercellular space (is) is greatly exaggerated over most regions of the cell, yet is maintained at desmosome sites. During the separation of the two cells, part of the spermatid plasma membrane has been detached. x 14,400. Fig. 15. Relationship of pachytene spermatocyte to Sertoli cell at ectoplasmic sites after treatment of tissue in hypertonic solution. The intercellular space (is) is greatly exaggerated at both ectoplasmic (arrowheads) and non-ectoplasmic sites. x 8700. Fig. 16. Survey micrograph of a Stage VI tubule after treatment in hypertonic solution. The Sertoli cell is markedly condensed and has separated from the spermatid cytoplasm in some areas (step 17), leaving an exaggerated intercellular space (is). In regions where ectoplasmic specializations (arrows) face the spermatid head, there is no artifactual separation of the two cells. x 21,500.

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their treatment with hypertonic solutions revealed that, in general, tissue preservation was poor. Sertoli cells were affected in two different and also opposite ways by this treatment. Most were reduced in size (condensed or shrunken) while a few were markedly swollen. Both types of artifacts provided useful information, and each will be described separately. Shrinkage of Sertoli cells and also germinal elements were evidenced by the increased density of the cells, and in many areas, an exaggeration of the intercellular space between two cell types (Figs. 1416). In regions where the two cells were facing each other, the intercellular space was examined to determine whether Sertoli and germ cells were readily pulled apart by the opposing forces generated during cell shrinkage. As a general rule, pachytene spermatocytes and early spermatids maintained close contact with Sertoli cells over about 50% of their surface area. The remaining surfaces were separated by spaces which were variable in width, some measuring up to several micrometers across. Pachytene spermatocytes and early spermatids were pulled apart from Sertoli cells at both ectoplasmic specialization sites as well as at non-ectoplasmic specialization sites (Fig. 15). Whether or not this artifactual separation occurred in any one particular area did not appear to be dependent on the presence or absence of ectoplasmic specializations. Although the two cells separated in areas where ectoplasmic specializations were present, they always remained in close apposition at desmosomelike sites. However, in many cases the germ

cell was fragmented (apparently from forces directly opposing one another) or torn away from the Sertoli cell at the periphery of the desmosomal contact (Fig. 14). The relationship of Sertoli cells to spermatids past step 7 of spermiogenesis was affected differently by the same procedure. These cells readily separated in areas lacking ectoplasmic specializations. However, in the head region where ectoplasmic specializations faced the acrosomal system, there was never an increase in the width of the intercellular space after this treatment procedure (Fig. 16). As mentioned above, a minority of Sertoli cells reacted to treatment in hypertonic solutions by becoming markedly swollen (Fig. 17). The plasma membranes of such cells maintained a normal relationship with nearby germ cells: e.g., similar to that seen in untreated tubules. The major difference between treated and untreated tissues was the density of the cytoplasmic matrix and the packing of organelles. In markedly swollen cells, the matrix displayed an ‘empty, watery appearance.’ Mitochondria, lysosomes, filaments, microtubules, etc. (Fig. 17) were sparsely scattered throughout the matrix of the cell. The relationship of these swollen Sertoli cells to elongating and maturing spermatids was examined. It was found that the ectoplasmic specializations remained undisturbed in their positions facing the acrosomes of elongated spermatids: i.e., the spacing of Sertoli plasma membrane. filaments and endoplasmic reticulum remained about the same as in untreated tissue. One minor difference was noted. In

Fig. 17-23. Sertoli-spermatid relationships after treatment of tissu-s in hypertonic solution. The Sertoli plasma membrane (large isolated arrows) is related to the spermatid by a normal spacing over both ectoplasmic and non-ectoplasmic sites (Fig. 17). The filamentous (asterisks) and cisternal comoonents (er) of the soecialization are likewise in their normal relationship to the spermatid head f in some regions, however, the cisternae have broken up into smaller vesicles (v). The remainder of the Sertoli organelles are scattered throughout an electron-translucent matrix (especially prominent in Figs. 17-19). Some microtubules (mt), seen within 200 A of the vesicles of the endoplasmic reticulum, are linked to them by short ‘bridges’ (arrowheads, Fig. 17, 19-23). Filaments of the specialization (small arrows) appear to either be inserted into the endoplasmic reticulum (Fig. 18) or into the Sertoli plasma membrane (Figs. 19. 20, 22). Fig. 17, x61.000; Figs. 19 and 21-23, x 140,000; Fig. 20. x 110,000.

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treated tissue, endoplasmic reticulum which was previously in the form of a single cisterna usually broke up and reformed as a row of small vesicles (Figs. 16-23). The absence of a dense background matrix within the Sertoli cells allowed for better visualization of structures near the surfaces of these cells. The filamentous component and the more deeply placed endoplasmic reticulum stood out in sharp contrast to the electron translucent background. Once again the filaments were measured and found to be 70-90 8, across. In many cases attachments of the filaments to the cisternae of the endoplasmic reticulum or to the Sertoli cell plasma membrane could be demonstrated (Figs. 1S-20). Microtubules were seen throughout the Sertoli cytoplasm and often found in the vicinity of the vesicles of endoplasmic reticulum (Fig. 17). In many instances where a microtubule was about 150-250 8, deep to the complex, there appeared to be a short bridge or link between the microtubule and the vesicle of the endoplasmic reticulum (Figs. 19-23). Having first been observed in swollen cells, these links were subsequently seen in untreated tissues, although they were most prominent in cells showing the swelling artifact.

Discussion As indicated in the introductory section of this report, it is generally believed that Sertoli ectoplasmic specializations are attachment devices between Sertoli and germ cells. The term that has recently been used to designate these structures, ‘junctional specialization’, implies that they must in some way act as a junction (Ross and Dobler, 1975; Ross, 1976, 1977). On the contrary, in the relationship of ectoplasmic specializations (filaments and endoplasmic reticulum) to germ cells, the former have never been shown to be a component of a junction between the two cells nor have they been shown to participate in attaching the two cells to one another. Although junctions which attach the two cells together may at some time in the future be shown to be present between Sertoli cells and germ cells (at ectoplasmic sites), it should not be assumed that junctions and ectoplasmic

specializations are structurally or functionally related. Most workers agree that a junction of the classical sense (Farquhar and Palade, 1963) is not present between Sertoli and germ cells at ectoplasmic sites. However, Connell (1976) has indicated in the dog that septate junctions are seen at these sites. The present investigator has not observed septate or septate-like junctions in thin sectioned material of the rat testis. The freeze-cleave method was used by Fawcett (1974) to examine plasma membranes within seminiferous tubules. He found, in a careful search of extensive areas, that there were no membrane specializations that would indicate either tight, gap, desmosome, or any other variety of junction between Sertoli cells and germ cells.* It seems appropriate to discontinue the use of the term junctional specialization in light of these negative findings. It is suggested that the more descriptive term ectopfasmic specialization be employed to describe the surface modifications of the Sertoli ceiis which face some germ cells. With regard to cell surface modifications at the level of the blood-testis barrier that are similar in appearance to the ectoplasmic specializations facing some germ cell types, it is also suggested that the terms junctional specialization or junctional complex (Dym and Fawcett, 1970) be dropped-again in favor of the term ectoplasmic specialization. Although there are numerous junctions of the tight, gap, and desmosome variety in this region, there is no evidence that Sertoli ectoplasmic specializations are part of this complex. The term junctional complex should be used only to describe this particular variety of junctions apart from an underlying ectoplasmic specialization. This investigator has observed (unpublished) that tight, gap, and desmosome junctions frequently occur between adjacent Sertoli cells, yet overlying subsurface modifications may be absent. The concept that ectoplasmic specializations and junctional complexes can exist as two separate morphologic and functional entities should be recognized, and the terminology should likewise reflect this difference. * What form the desmosome-like junctions (present at non-ectoplasmic sites) described by Russell (1977a) might take in freeze-cleave preparations has yet to be determined.

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It was assumed by this author that junctions which possess the property of cell-tocell adherence should retain this property even after treatment with mildly hypertonic solutions (Russell, 1977a). The forces which pull cells apart (as a result of cell shrinkage) were quite capable of separating pachytene spermatocytes and early spermatids from Sertoli cells regardless of whether there was ectoplasmic specialization present. Thus, it was felt that these two cell types were neither attached by a junction (as were desmosomelike sites) nor held together mechanically. The head regions of cells more advanced than step 8 maintained the close spacing to Sertoli ectoplasmic specializations (after cytoplasmic condensation with hypertonic solutions), whereas the trailing cytoplasmic masses readily separated from the Sertoli cells. These features indicated that the two cells were bound together in some way at ectoplasmic specialization sites. Since material was not seen traversing the intercellular space (which would have indicated a junction of some type), it was assumed that the ectoplasmic specializations were capable of maintaining a grasp on the heads of the spermatids. Ross and Dobler (1975) have suggested that the ectoplasmic specializations, seen at the level of the blood-testis barrier, become attached to spermatocytes as the latter cells pass toward the adluminal compartment. Once this has occurred, the cells would remain attached by specializations until the time of spermiation. The present study and that of Russell (1977b) have indicated, after a careful search of extensive areas of the Sertoli ectoplasmic zones which surround leptotene, zygotene and young pachytene cells, that these regions are never seen to display ectoplasmic specializations facing (in apposition to) the aforementioned germ cells. It was shown (Russell, 1977b) that Sertoli-Sertoli ectoplasmic specializations at the level of the blood-testis barrier are resorbed to allow passage of leptotene spermatocytes across the barrier: prior to this event, new specializations have formed above the spermatocytes. Such findings would indicate that Sertoli-Sertoli ectoplasmic specializations are separate entities and independent of those specializations which are seen facing germ cells. These findings have emphasized the

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relationship of Sertoli ectoplasmic specializations to developing germ cells. The first appearance of these structures (in relationship to germ cells) during the spermatogenic cycle was in apposition to mid-pachytene spermatocytes in late Stage VII and early Stage VIII. Throughout most of the remainder of the spermatogenic process, they remained in apposition to germ cells. A shift from this position occurred in Stage VII (exactly two cycles after their initial appearance), as the spermatids (step 19) approached release. Once displaced, the ectoplasmic specializations were seen along the Sertoli cells at their free surfaces or in apposition to residual bodies. They were also seen near, and in partial relationship to, the step 7 spermatids and pachytene spermatocytes of late Stage VII and Stage VIII. The series of observations just described suggest that ectoplasmic specializations which are related to germ cells are reutilized in a cyclic manner. The process just described is diagrammed in Fig. 24. The essential steps in this process include: (1) appearance of ectoplasmic specializations facing mid-pachytene spermatocytes in Stage VII (Fig. 24a); (2) association of ectoplasmic specializations with germ cells until step 19 of spermiogenesis (Stage VII; Fig. 24b, c, d); (3) shift of specializations from their positions facing step 19 spermatids and appearance of specializations on the free border of the Sertoli cells and in apposition to the residual cytoplasm (Fig. 24e); (4) movement of specializations to positions facing the earlier generation of step 7 spermatids (Stage VII) and pachytene spermatocytes (of Stage VII; Fig. 24f). One mechanism by which the transfer of Sertoli ectoplasmic specializations in late Stage VII (from a position near the lumen to one considerably nearer the base of the tubule) could occur might involve a flow of Sertoli cytoplasm (toward the base of the tubule) that would bring them into apposition to spermatids and pachytene spermatocytes of Stage VII. Another event occurring at the same time indicates that this cytoplasmic flow is indeed taking place. The apical Sertoli expansions which are holding the late spermatids into the tubular lumen are gradually withdrawn towards the base of the tubule in late Stage VII and Stage VIII, eventually leaving the late spermatids free in the lumen (Russell and Clermont, 1976).

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Microtubules are abundant in these processes (unpublished observations) and may be the primary structure which influences the remodeling of the Sertoli cell. Thus, at this particular time before sperm release, there must be flow of Sertoli cytoplasm towards the base of the tubule, which would account for the observed movement of Sertoli ectoplasmic specializations along this direction. During and following the meiotic divisions, the number of cells exposed to Sertoli ectoplasmic specializations appeared substantially reduced, such that the specialization was only occasionally seen in sections through young spermatids. With the production of four spermatids from one primary spermatocyte and the resultant increase in surface area (displayed by these new cells), it was not surprising that the number of cells (in sections) displaying this specialization appeared diminished. Nevertheless, it is probably true that the absolute amount of specialization present during meiotic divisions and up to late step 7 of spermiogenesis was unchanged. By Stage VIII of the next cycle, the step 8 spermatids were exposed to

considerably more extensive areas of Sertoli ectoplasmic specialization. This occurred at a time when the older generation of spermatids were approaching release into the tubular lumen. The shift in position of Sertoli ectoplasmic specializations away from the late spermatids explains not only how midpachytene spermatocytes of late Stage Vi1 and Stage VIII come to be the first cells in the spermatogenic process to face these structures, but also why step 8 spermatids displayed considerably more extensive areas of specialization facing their cytoplasmic boundaries (than did early spermatids; Fig. 24e). Pre-existing specializations seen facing the early generation of spermatids (steps l-7), combined with those specializations cast off from the late generation of spermatids (step 19), would both become associated with the step 8 cells. As the elongation phase of spermiogenesis proceeded and the acrosome spread out over the spermatid head, it was apparent that the absolute amount of Sertoli ectoplasmic specialization apposing this surface of the spermatid must have also increased. The possibility of de nova synthesis of

Fig. 24. Diagrams illustrating the relationships of Sertoli cells (shaded) to germ cells at ectoplasmic specializations (arrowheads) during the spermatogenic cycle. The stages of the cycle are listed below each diagram. (a) initial appearance of ectoplasmic specializations The most immature germ cell seen facing ectoplasmic specializations is the Stage VII (late) pachytene spermatocyte.Aspermatogonium is seen at the base of the tubule. Sertoli-Sertoli ectoplasmic specializations are indicated by arrows (b) presence of Sertoli ectoplasmic specializations facing a newly formed spermatid. Most of the spermatid is surrounded by small veil-like processes of Sertoli cytoplasm (c) appearance of extensive areas of Sertoli ectoplasmic specialization facing the acrosome of a step 8 spermatid. Sertoli recesses which contain the heads of the spermatids are in evidence (d) increase in surface area of the spermatid head and acrosome with a parallel increase in surface area of the apposing ectoplasmic specialization and a deepening of the Sertoli recess (e) shift in position of ectoplasmic specializations in Stage VII, leaving the spermatid head free of these structures. The specializations are now seen on the free surfaces of the Sertoli expansion and in the Sertoli connecting stalk (f) appearance of ectoplasmic specializations in apposition to the earlier generation of spermatids (late step 7 and early step 8) and pachytene spermatocytes of late Stage VII and early Stage VIII. Sertoli recesses around the spermatid heads are no longer in evidence.

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ectoplasmic specialization at this time should not be ruled out. It is not difficult to envision that with time, and without some synthesis, this peculiar structure would be lost as the result of imperfect recycling. Concomitant with the increase in surface area which was manifested by the Sertoli ectoplasmic specializations in Stage VIII, the spermatid heads (step 8) became situated within recesses of the Sertoli cells. As the surface area of the ectoplasmic specializations increased (paralleling the increase in surface area of the acrosomes as they spread over the nuclei), these recesses deepened and the heads of the spermatids became positioned nearer to the bases of the Sertoli cells at a level between many pachytene spermatocytes. During the period that the ectoplasmic specializations faced the acrosomalsystems of the spermatids (about 12 days, Clermont et a[., 1959), these recesses were in evidence. As the spermatids approached release into the tubular lumen, the Sertoli ectoplasmic specializations shifted from their characteristic position facing the acrosomes of the spermatids. Shortly thereafter, the recesses in which the heads of the spermatids were embedded were ‘erased’. The development of Sertoli recesses shortly after the appearance of extensive areas of Sertoli ectoplasmic specializations (facing the heads of spermatids) suggests that there is a relationship between the two events. It is postulated that the Sertoli ectoplasmic specializations sufficiently stiffen the surface aspects of the Sertoli cells such that they closely conform to the contour of the spermatid heads and thus provide recesses for the head regions of these cells. Once formed, the recesses were not erased until after the Sertoli ectoplasmic specializations had lost their positions facing the late spermatids. Approximately one day later and after the dissolution of another type of holdfast device (formed in late Stage VI), the tubulobulbar complex (Russell and Clermont, 1976), the spermatids were released. Since desmosome-like structures are only rarely seen attaching Sertoli cells to elongating and condensing spermatids (past step 9 of spermiogenesis), it is unclear how these spermatids might be prevented from premature release into the tubular lumen. Most investigators believe that Sertoli ectoplasmic

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specializations function in preventing premature release of elongating cells by binding the two cell types together (Nicander, 1967; Sapsford and Rae, 1969; Fawcett and Phillips, 1969; Ross and Dobler, 1975; Ross, 1976) or by grasping the spermatid heads (Toyama, 1976). The observations presented in this study suggest that the latter theory (grasping) may be important in explaining how these cells are prevented from being sloughed prematurely. Whether or not actin-like filaments in the Sertoli ectoplasmic specializations (Toyama, 1976) actually tighten in order to grasp the heads of the spermatids has yet to be determined, but the failure of elongating spermatids to separate away from ectoplasmic sites after treatment with hypertonic solutions supports this concept. It was shown that when a marked swelling of the Sertoli cell was provoked (using hypertonic fixatives), the components of the ectoplasmic specializations were maintained in their normal relationship to the elongated spermatids. Unlike other organelles which were widely scattered through the cytoplasm, the ectoplasmic specializations remained in a compact form, a feature which would indicate that the Sertoli plasma membrane, filament bundle, and endoplasmic reticulum were held together as a single unit. Indeed, connections between filaments and between Sertoli plasma membrane and cisternae of endoplasmic reticulum were found in both treated and untreated tissues. In addition, nearby microtubules were seen to be attached to the vesicles (cisternae) of the specializations by small bridges. The ectoplasmic specializations might thus be influenced, as a whole, by the ‘actions’ of microtubules. These observations may be useful to explain how spermatids might either be drawn to the base of the seminiferous tubule (as seen in early Stage II) or moved toward the lumen of the tubule (as seen in late Stage VI). Microtubules are generally thought to be important cytoskeletal elements which, in this case, are undoubtedly important in maintaining and also influencing the shape of the Sertoli cell. Although the observations of this report have been directed towards a better understanding of the nature and functioning of ectoplasmic specializations at the Sertoli-

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germ cell interface, they also may be of value in understanding the function of structures of similar appearance at the level of the blood-testis barrier. There does not appear to be a lack of junctions (desmosomes, gap, narrow, and tight) where two Sertoli ectoplasmic specializations are in juxtaposition. Some of these undoubtedly function to bind the two cells together. What purpose then might these ectoplasmic specializations serve? Dym and Fawcett (1970) demonstrated that a certain population of cells (spermatogonia and preleptotene spermatocytes) resides at the base of the tubule (basal compartment) and is separated from other more adluminal germ cells by intervening Sertoli cells (see Fig. 24a). The blood-testis barrier was described as being formed by tight junctions at sites where adjacent Sertoli cells meet (Dym and Fawcett, 1970; Ross, 1970). Germ cells of the basal compartment are seen in a position at the base of the tubule and between Sertoli cells. Those which become committed to the maturation process and become leptotene spermatocytes begin their transfer towards the adluminal compartment. This transfer involves the interaction of two adjoining Sertoli cells (Russell, 1977b) such that in their movement toward the lumen, the spermatocytes are always seen between adjacent Sertoli cells. In order for this process to take place, it is necessary that the maturing germ cells (connected by intercellular bridges) always be positioned between the larger adjoining Sertoli cells. If, for example, the basal aspect of one Sertoli cell were to completely cover over a germ cell, this latter cell would not be able to move towards the lumen (to complete its maturation process). Complex interdigitations of adjoining Sertoli cells would interfere with this movement, as well as with the development of the extensive tight junctional complex between Sertoli cells. Therefore, it seems important that there be some means by which the position and shape of the lateral aspects (at the base) of the Sertoli cells could be regulated. The ectoplasmic specializations of adjoining Sertoli cells might impart a certain rigidity to the membranes of these cells, assuring that their membranes remain relatively firm and in juxtaposition above the developing germ cells. Such rigid membranes would presum-

ably restrict cytoplasmic streaming, and thus, the cell would be resistant to major changes in configuration such as seen when complex interdigitations form between cells. The postulated functions of ectoplasmic specializations as given above, whether describing Sertoli-germ cell or SertoliSertoli interactions, embrace a common mechanism by which the ectoplasmic zone of Sertoli cells is made rigid by the ectoplasmic specializations. Presumably, only the filaments are important in this process, for it is difficult to envision how the overlying cisternae of endoplasmic reticulum could impart significant rigidity to the cell surface. Endoplasmic reticulum perhaps serves as a mantle to which structures such as microtubules or other cytoskeletal elements might be attached, thereby influencing the cell configuration and shape. However, the possibility that they also function in some yet undefined metabolic sense should not be ruled out. Summary

Endoplasmic reticulum and filaments at the subsurface aspect of the Sertoli cell comprise the ectoplasmic (‘junctional’) specialization: a structure of unknown function which is often seen facing germ cells. The relationship of Sertoli ectoplasmic specializations to developing germ cells was studied in the rat at all stages of the cycle (classification of Leblond and Clermont, 1952). The most immature germ cell types which faced Sertoli extoplasmic specializations were the pachytene spermatocytes of Stage VII. In fact, all germ cell types more advanced than these cells (up to and including late spermatidsstep 19) were seen facing these specializations. Morphological data were accumulated which indicated that Sertoli ectoplasmic specializations are reutilized, being transferred from late spermatids (step 19) of Stage VII to the more immature pachytene spermatocytes and cap-phase spermatids of late Stage VII. No evidence was found to show that ectoplasmic specializations were junctions in any classical sense (Farquhar and Palade, 1963); thus, it was suggested that the term ‘junctional specialization’ be dropped in favor of the term ectoplasmic specialization. Data obtained from tissues treated with hypertonic solutions showed that

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spermatids less advanced than step 7 could easily be pulled away from Sertoli cells at ectoplasmic specialization sites, whereas those past step 7 never separated at ectoplasmic sites. Ectoplasmic specializations became well developed in cells past step 7 of spermiogenesis. At steps 8 through 19, it appeared that the subsurface filaments were important in maintaining a firm grasp on the spermatid heads. During steps 8 and 9 the spermatids began to elongate and were seen within recesses in the Sertoli cell. Throughout most of the remainder of spermiogenesis, these recesses were maintained. However, soon after the ectoplasmic specializations shifted from their position facing the late (step 19) spermatids, the recesses were obliterated. The spermatids were released shortly thereafter. It is postulated that ectoplasmic specializations associated with spermatids more advanced than step 7 function in imparting rigidity to the surface of the Sertoli cell, providing a recess to contain the

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spermatid during its elongation and maturation phases of development. Obliteration of this recess would facilitate late spermatid release. Attachment of microtubules of the Sertoli cell to the overlying specializations was demonstrated, thus providing morphological evidence to suggest the involvement of the Sertoli cell in the observed movements of spermatids either toward the base (in Stage II) or toward the surface of the seminiferous tubule (in late Stage VI). The function of ectoplasmic specializations of similar appearance, at the interface of adjoining Sertoli cells, is discussed in relation to the above findings. Acknowledgements The author gratefully acknowledges the technical assistance of MS Beryn Frank. This work was supported by Grant NIH-HD 10266-01 from the National Institute of Child Health and Human Development.

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FAWCETT, D. W. 1970. Interrelations of cell types within the seminiferous epithelium and their implications for control of spermatogenesis. In: The Regulation of Mamm&an Reproduction (eds. S. Segal, R. Grozier, P. Corfman, P. Condliffe). C. C. Thomas Pub., Springfield, Ill., pp. 91-99. FAWCETT, D. W. 1974. Observations on the organization of the interstitial tissue of the testis and on the occluding cell junctions in the seminiferous epithelium. Schering Symp. on Contraception-Male. Advances in Bioscience, 10, Pergamon Press, Oxford. FAWCETT, D. W., LEAK, L. V. and HEIDGER, P. M. 1970. Electron microscopic observations on the structural components of the blood-testis barrier. J. Reprod. Fert., Suppl., 10, 105-122. FAWCETT, D. W. and PHILLIPS, D. M. 1969. Observations on the release of spermatozoa and on changes in the head during passage through the epididymus. J. Reprod. Ferr., Suppl., 6, 405-418. FLICKINGER, C. J. 1967. The postnatal development of Sertoli cells in the mouse. Z. Zellforsch. mikrosk. Anal., 78, 92-l 13.

FLICKINGER, C. and FAWCETT, D. W. 1967. The junctional specializations of Sertoli cells in the seminiferous epithelium. Anat. Rec., 158, 207-221. GRAVIS, C. J., YATES, R. D. and 1-1~1CHEN. 1976. Light and electron microscopic localization of ATPase in normal and degenerating testes of Syrian hamsters. Am. J. Anar., 147, 419432. LEBLOND, C. P. and CLERMONT,Y. 1952. Definition of the stages of the cycle of the seminiferous epithelium in the rat. Ann. N. Y. Acad., Sci. 55, 548-573. NICANDER,L. 1963. Some ultrastructural features of mammalian Sertoli cells. J. ufrasfruct. Res., 8, 19%191. NICANDER, L. 1967. An electron microscopical study of cell contacts in the seminiferous tubules of some mammals. Z. Zellforsch. microsk. Anat., 83, 375-397. Ross, M. H. 1970. The Sertoli cell and the blood-testicular barrier: An electron-microscopic study. Fortschritte der Andrologie [Morphological Aspects of Andrology] (eds. A. F. Holstein and E. Horstmann), pp. 183-186. Ross, M. H. 1976. The Sertoli cell junctional specializations during spermiogenesis and at spermiation. Anat. Rec., 186, 74-104. Ross, M. H. 1977. Sertoli-Sertoli junctions and Sertoli-spermatid junctions after efferent ductule ligation and lanthanum treatment. Am. J. Anot., 148,49-56. Ross, M. H. and DOBLER, J. 1975. The Sertoli cell junctional specializations and their relationship to the germinal epithelium as observed after efferent ductule ligation. Anar. Rec., 183, 267-292. RUSSELL,L. D. 1977a. Desmosome-like junctions between Sertoli ceils and germ cells in the rat testis. Am. J. Anat., 148, 301-312. RUSSELL, L. D. 1977b. Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am. J. Annt., 148, 313-328.

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RUSSELL, L. D. 1977~. Membranous structures within nuclei of spermatocytes in the adult male rat. Biol. Reprod. (in press). RUSSELL, L. D. and CLERMONT,Y. 1976. Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules. Amt. Rec., 187, 347-366. SAPSFORD, C. S. and RAE, C. A. 1969. Ultrastructural studies on Sertoli cells and spermatids in the bandicoot and ram during the movement of mature spermatids into the lumen of the seminiferous tubule. Amt. J. Zool., 17, 415445. TOYAMA, Y. 1976. Actin-like filaments in the Sertoli cell junctional specializations in the swine and mouse testis. Amt. Rec., 186, 477491. VITALE, R., FAWCETT, D. W. and DYM, M. 1973. The normal development of the blood&estis barrier and the effects of clomiphene and estrogen treatment. Anaf. Rec., 176, 333-344.