Further observations on tubulobulbar complexes formed by late spermatids and sertoli cells in the rat testis.код для вставкиСкачать
Further Observations on Tubulobul bar Complexes Formed by Late Spermatids and Sertoli Cells in the Rat Testis LONNIE D. RUSSELL Department of Physiology and School of Medicine, Southern Illinois University at Carbondale, Carbondale, Illinois 62901 ABSTRACT The present study extends earlier findings (Russell and Clermont, '76) by describing additional details of the structure, relationships, distribution, origin, and fate of tubulobulbar complexes. Freeze-fracture and thin section observations reveal few membrane associated particles and no appreciable cell coat in the membranes forming the tubulobulbar complex. The opposing plasma membranes of the complex do not appear to form a junction, although junctional formation might be expected by the close proximity (4.0 nm apart) of the Sertoli and spermatid plasma membranes. Sertoli filaments of the network which encircle the tubular portion of the complex measure 5.0-7.0 nm across and appear to insert into the Sertoli plasma membrane. Tubulobulbar complexes initially form in association with a cell surface modification (bristlecoated pit) of the Sertoli plasma membrane. The first tubulobulbar complexes develop large bulbous components (up to 2.5 p m across), which soon lose connection with the spermatid and become incorporated into large phagocytic vacuoles (secondary lysosomes). As bulbs undergo dissolution, newly formed tubulobulbar complexes are observed to replace these structures. Thus, the data indicate that more than one generation of tubulobulbar complexes develop. Moreover, the tubular and bulbous portions of most dissociating complexes (as well as neighboring Sertoli lysosomes) show acid phosphatase activity. Near the time of sperm release, all complexes a t the concave aspect of the spermatid head are resorbed. New tubulobulbar complexes, many lacking terminal bulbous dilations, form a t the dorsal and lateral aspects of the spermatid head. These persist even after all ectoplasmic specializations and most Sertoli cytoplasm have been withdrawn from a position facing the spermatid head. The presence of tubulobulbar complexes just prior to the time of sperm release is in support of previous findings indicating that tubulobulbar complexes participate in anchoring the head of the late spermatid prior to sperm release. Past the zone of sperm release a few abnormally shaped cells, which had not been released with other spermatids of the same generation, display intact tubulobulbar complexes. The persistence of these and other structures may be the means by which abnormally shaped spermatids are retained and prevented from traversing the male duct system. The tubulobulbar complex has been described as a structural relationship between Sertoli cells and late spermatids as the latter cells approached release into the tubular lumen (Russell and Clermont, '76; also fig. 1). The postulated function of tubulobulbar complexes is to act as anchoring devices which retain the heads of the spermatids a t the surface of the seminiferous epithelium. DissoluANAT. REC. (1979)194: 213-232. tion of these structures would contribute to the release of the spermatozoan from the seminiferous epithelium into the tubular lumen (spermiation). The present study provides new data concerning tubulobulbar complexes and the interrelationships of Sertoli cells. It emphasizes the progressive turnover Received June 8, '78. Accepted Nov. 21,'78. 213 214 LONNIE D. RUSSELL of tubulobulbar complexes in the period prior to sperm release and also the role of the Sertoli cell in this process. MATERIALS AND METHODS Preparation of freeze-cleave replicas Four Sprague-Dawley rats were anesthetized with sodium pentobarbital and the testes fixed by vascular perfusion (Vitale et al., ’73) with a 3.5%solution of glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.4) preceded by a washout of 0.9% saline. After the testes hardened (-20 minutes) they were removed, diced into 1.0-mm blocks and placed in three changes of a 30%glycerol solution. The tissue was frozen in freon, cooled with liquid nitrogen, and fractured in a Balzers freeze- fracture apparatus. Platinum replicas coated with carbon were prepared, isolated and viewed under a Phillips 201 electron microscope. The terminology employed to describe membrane faces was that proposed by Branton et al. (‘75). Standard tissue preparation Testes from two Sherman and four SpragueDawley rats were fixed by vascular perfusion (see above) of 5.0%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Testes were removed, cut into small pieces and fixed in perfusate for an additional hour. Tissue was washed in buffer and post-fixed either with 2.0% osmium tetroxide, or with a 2.0% osmium:2.5% potassium ferrocyanide mixture A b breuiations b, Sertoli plasma membrane b , spermatid plasma membrane a, acrosome b, bulbous portion of the tubulobulbar complex cp, bristle-coated pit es, ectoplasmic specialization Es, extracellular face of the Sertoli plasma membrane Et, extracellular face of the spermatid plasma membrane f, filaments 1, lysosome n, nucleus Ps, protoplasmic face of the Sertoli plasma membrane Pt, protoplasmic face of the spermatid plasma membrane pv, phagocytic vacuole S, Sertoli cell t, tubular portion of the tubulobulbar complex ser, smooth endoplasmic reticulum Fig. 1 The relationship of the late (step 19) spermatid to the Sertoli cell apical expansion a t the luminal aspect of the seminiferous tubule is depicted. In this micrograph, a tubulobulbar complex, showing both tubular and bulbous portions, is seen in longitudinal profile. The perinuclear cytoplasm (pc) of the spermatid head is continuous with the cytoplasm of the tubular portion of the complex. In this particular micrograph this body of cytoplasm appears somewhat denser than is usually seen. The intercellular space between spermatid and Sertoli cell (S) is considerably reduced in the region where these two cells form the tubulobulbar complex. Facing the head of the spermatid is a subsurface modification of the Sertoli cell, consisting of filaments and more deeply placed saccules of endoplasmic reticulum, termed the ectoplasmic specialization (Russell, ’7%; es). Tissue treated with a ferrocyanide:osmium mixture. x 39,000. Figs. 2-4 Tissues treated with an osmium:ferrocyanide mixture. Mid-Stage VII. Fig. 2 Longitudinal section of a tubulobulbar complex measuring approximately 4.0 wm in length. The diameter of the tubular portion of the complex is remarkably uniform. In this particular micrograph, the bulb appears to be unusually small. X 50,000. Fig. 3 Cross section of the tubular portion of the complex showing the trilaminar appearance of both spermatid and Sertoli plasma membranes. The intercellular space is approximately one half the width of one of the individual plasma membranes. No evidence of a glycocalyx is seen on the external aspect of either membrane. Filaments around the tube are poorly visualized. x 165,000. Fig. 4 Longitudinal section of a portion of tubulobulbar complex showing nearby Sertoli filaments. In several areas (indicated by arrows) these filaments appear to make contact with the Sertoli plasma membrane. x 83,000. Figs. 5-7 Tissues secondarily treated with osmium. Mid- and late-Stage VII. Fig. 5 Longitudinal section of the tubular portion of a tubulobulbar complex of late-stage VII showing the “wavy” course which the complex often takes. Sertoli filaments are prominent and extend about 0.2-0.3p m into the Sertoli cell. These filaments are packed most tightly near the Sertoli plasma membrane. In a few areas (arrow) the spermatid tube is discontinuous. The Sertoli cytoplasm near the tube (also fig. 2) is virtually free of organelles. x 36,000. Fig. 6 Cross section of a spermatid showing only the ventro-lateral aspect of the head. Continuity of the plasma membranes of the Sertoli cell and spermatid with their respective membranes forming the tubular portion of the complex is demonstrated. The plasma membrane of spermatid head proper appears thicker than it is within the tubular portion of the complex. This is the result of the presence of an appreciable cell-coat on the spermatid plasma membrane of the head, whereas this feature is not evident in the tube. X 65,000. Fig. 7 Cross-section of the tubular portion of the tubulobulbar complex. Although filaments are prominent, the trilaminar structure of membranes is not evident. The intercellular space in tissues treated in this manner appears slightly wider than that shown in figure 3. x 90,000. 216 LONNIE D. RUSSELL (Russell and Burguet, ’77; modified from Karnovsky, ’71). Subsequent dehydration, infiltration and embedding in Epon were according to standard techniques. At the time of embedding, seminiferous tubules were oriented to produce either longitudinal or crosssectional profiles upon sectioning. Gray, silver and silver-gold appearing thin sections were prepared with an ultramicrotome, stained with uranyl acetate and lead citrate and examined with a Phillips 201 transmission electron microscope. tissues incubated in medium lacking substrate or with the addition of sodium fluoride. RESULTS Descriptive data All membranes participating in the formation of the tubular or bulbous portions of the tubulobulbar complex measured about 7.5 nm across. The spermatid plasma membrane over most other regions of the head appeared thicker (about 14 nm; figs. 1, 6, 7; Nicander, ’67), but this appearance was due to the presence of a substantial glycocalyx which was not Timing of spermatogenesis observed in the membrane of the tubulobulbar The classification of Leblond and Clermont complex. The evaginated process of the sper(’52) was used to select tubules a t the desired matid which forms the tube appeared much stages of the spermatogenic cycle. Since it closer to the Sertoli plasma membrane (fig. 3) was necessary to follow sequential events re- than reported for osmium treated tissues lating to the formation and degradation of t u - (Russell and Clermont, ’76). Thus, in ferrobulobulbar complexes, it was critical that cyanide-osmium treated tissues the interStages VII and VIII be divided into recogniza- cellular space is recorded to be about 4.0 nm in ble “substages” which could be used to “time” width whereas by osmium-only treatment it related events. The term substage is used to was about 6.0 nm (compare figs. 3, 7). Sertoli designate a particular cross-sectional profile filaments surrounding the tubular portion of of a seminiferous tubular subsegment. These the complex were not as conspicuous after fersubstages, when placed in a logical time rocyanide:osmium treatment (figs. 2, 3) but sequence order, could then be selected for ob- were more prominent following standard gluservation a t the electron microscope level. The taraldehyde-osmium treatment (figs. 5, 7). classification of Perey et al. (‘61) based on The degree of packing of the network of filastudies of longitudinal sections of seminifer- ments was tightest just deep to the Sertoli ous tubules and previously defined subseg- plasma membrane (figs. 5, 7, 8). Isolated filaments was used in the designation of Stage ments measured 5.0-7.0 nm and in many secVII into early, middle and late substages. Our own observations and those of Perey et al. Fig. 8 Fortuitous longitudinal section through a were used to divide Stage VIII into three tubulobulbar complex showing both the proximal (long substages. In early Stage VIII the apical Ser- arrow) and distal (short arrow) tube and an intervening The distal tube faces a Sertoli bristle-coated pit. toli expansion was seen to engulf the whole bulb. X 30,000. head region where as in mid-Stage VIII it was Fig. 9 The distal aspect of a tubulobulbar complex, only related to a small area of t h e head portion similar to t h a t shown in figure 8, is seen a t high magof this cell (Russell, ’77b).Late Stage VIII was nification. A bristle-coated pit is present a t this site and can be identified by t h e fuzzy substance projecting from designated as the particular substage in t h e internal aspect of t h e Sertoli plasma membrane. The which sperm release had already taken place spermatid tube is slightly expanded a t its distal tip. Fibrils are seen extending from i t to the Sertoli bristle(Perey et al., ’61). Acid phosphatase cytochemistry Testes were perfused with a 2.0% buffered glutaraldehyde, washed in buffer and slices of tissue (75-100 p m in thickness) were incubated at 37°C in a medium containing sodium-/3-glycerophosphate a t pH 5.0 for 90 minutes (Barka and Anderson, ’63). Tissues were washed in buffer containing sucrose and post-fixed in 1.0%osmium tetroxide. Dehydration, infiltration and embedding were similar to that described above. Controls consisted of coated pit. X 200,000, Fig. 10 This large bulbous component of a tubulobulbar complex is typical of those seen in early Stage VII. I t measures approximately 2.0 p m by 1.0 pm. Dispersed flocculent material is observed inside t h e bulb and smooth endoplasmic reticulum of the Sertoli cell is closely apposed to it. X 70,000. Fig. 11 High magnification micrograph of the limiting aspect of a bulbous portion of the tubulobulhar complex. The spermatid and Sertoli plasma membranes are about 4.0 nm apart and neither exhibit a glycocalyx. In thin sections of this type, there is no evidence of junctional formation between the two cells. Smooth endoplasmic reticulum is positioned about 15.0 nm from t h e Sertoli plasma membrane. X 185,000. TUBULOBULBAR COMPLEXES OF THE RAT 217 218 LONNIE D. RUSSELL tions appeared to make contact with the Sertoli plasma membrane (fig. 4). In longitudinal section the tubular portion of the complex was rarely linear, but usually was seen to describe a wavy course which took it in and out of the plane of section (figs. 2, 5). The length of the entire complex was variable, most commonly measuring 3.0-4.0p m and in some cases up to 5.0 p m (fig. 5). In all areas of the tubular portion of the complex, the diameter of the spermatid tube and width of the intercellular space were remarkably uniform (figs. 2-81. The bulbous portion of the tubulobulbar complex also displayed a 4.0 nm intercellular space (fig. 11).Individual bulbs measured up to 2.5 p m in diameter although the majority were less than 1 p m across. Most bulbs contained evenly dispersed flocculent material (figs. 8, 101, and a few bulbs showed a concentration of dense homogeneous substance (fig. 23). In some sections it was noted that the distal portion of bulb narrowed down to form tubular structure similar to t h e tube connecting the bulb to the spermatid head proper (fig. 8). This distal spermatid tube extended only a short distance (0.1-0.5 pm), and at its termination the facing Sertoli cytoplasm invariably displayed a bristle-coated pit (figs. 8, 9). Freeze-cleaue observations Replicas from Stage VII and VIII tubules (Stages in which tubulobulbar complexes are present) could be recognized by identifying the early generation of spermatids (steps 7 and 8) as well as the large fully-formed flagella of the late generation of spermatids. Tubulobulbar complexes were encountered within a recognizable apical Sertoli expansion. The characteristic form of the head of the late spermatid was also evident (figs. 12, 13). Only the Sertoli plasma membranes which surrounded the spermatid evagination was fractured in the plane of the membrane; however, in the cluster of bulbs an occasional area where the fracture plane coursed through the spermatid plasma membrane could be detected (figs. 12-14). In all of the membrane faces studied, it was apparent that these membranes displayed only an occasional membrane associated particle. In fact, none were seen in the tubular portion of the complex. For comparison, figure 16 shows intracytoplasmic vesicles within a nearby step 7 spermatid of the same stage, all of which display numerous membrane associated particles. A time-course study of tubulobulbar complexes Early stage VII Formation of tubulobulbar complexes in large numbers takes place a t the beginning of Stage VII. Prior to this time (in Stage VI) the heads of the late spermatids are deeply embedded within Sertoli recesses and only rarely during this period were these complexes observed. The newly formed complexes could be described as short (0.1-0.2p m ) tubular projections of the spermatid which face a corresponding indentation of the Sertoli cell (fig. 17). In every profile of this type observed, the spermatid projection was seen to oppose a bristle-coated pit in the depth of the Sertoli recess. (Coated pits are frequently seen a t the surface of the Sertoli cell including that area of plasma membrane which faces the spermatid head.) Sertoli filaments were usually found around the invaginated Sertoli plasma membrane (fig. 17). Figs. 12-16 Freeze-fracture replicas of step 19 spermatids, Sertoli cells, and tubulobulbar complexes formed by the two cells. All figures have been oriented such that the direction of platinum deposition is from the bottom to the top of the figure (with the exception of fig. 14 which has been shadowed from the top). Figs. 12, 13 The spermatid head (sh) is cross-fractured and the apical Sertoli expansion occupies most of the area in the figure. Fracture faces indicated are as follows: E face of the Sertoli plasma membrane around the head; P and E faces of the Sertoli plasma membrane of the bulb; and P and E faces of the spermatid plasma membrane of the bulb. The tubular portion of the complex can be identified (isolated arrow) as well as the ectoplasmic specialization, and smooth endoplasmic reticulum which faces the bulb. Numerous membrane associated particles are present in the Sertoli plasma membrane facing the head (fig. 12), yet few are seen in the bulbous portion of the complex (figs. 13, 14). X 29,000;X 42,000. Fig. 14 Replica showing the apical Sertoli expansion in the region of the clustered bulbs. Indicated are likely areas of the P and E fracture faces of the Sertoli plasma membrane in both the tube and bulb; and the P and E face of the spermatid plasma membrane. Membrane associated particles are not seen on the membranes forming the bulb. x 37,000. Fig. 15 Longitudinal fracture of the tubular portion of the complex showing predominantly the P and E faces of the Sertoli plasma membrane. The spermatid tube is also visible (isolated arrow). The membranes display a slightly “lumpy” appearance (as do all membranes of this area) but there are no particles intercalated within them. X 60,000. Fig. 16 This cross-fracture of a step 7 spermatid shows both nuclear and cytoplasmic areas of the cell. Unlike tubulobulbar complexes, vesicles of the cell demonstrate numerous membrane associated particles. The chromatoid body (c) is also fractured, and it is shown to be composed of closely aggregated cytoplasmic particles associated with small cytoplasmic vesicles. x 39,000. TUBULOBULBAR COMPLEXES OF THE RAT 219 220 LONNIE D. RUSSELL A bulbous structure was evident as part of complexes which were slightly larger than those described above. This dilated component apparently developed somewhere in the midregion of the spermatid tube since constricted spermatid tubes were seen both proximal and distal to the dilation (fig. 18). At this time, Sertoli endoplasmic reticulum of the smooth variety was generally observed facing the bulb (fig. 18). In slightly more developed profiles, the bulbs were quite large, measuring up to 2.5 p m across (fig. 10). The proximal tubular portion of the complex elongated and by early Stage VII attained a length of 3.0-5.0 p m (fig. 2). Concomitant with the formation of tubulobulbar complexes in early Stage VII, a remodeling of the Sertoli cell cytoplasm took place. An apical Sertoli expansion circumscribing the spermatid head was formed (figs. 19; Russell and Clermont, ’76) and the head was noticeably more sickle-shaped. Thus, tubulobulbar complexes emanating from the concave aspect of the spermatid ended in bulbous expansions which were clustered together among lysosomes and lipid droplets. Tubulobulbar complexes also formed a t the lateral or the dorsal convex aspect of the spermatid (fig. 171, although a t this time they were not commonly found to emanate from these sites. Near the completion of early Stage VII, it was observed that not all bulbous endings appeared identical. One type of bulb appeared similar to those which initially formed in early Stage VII (fig. 19). In another type, the spermatid plasma membrane was found to be lacking a connection with the spermatid head, i.e., in numerous sections and profiles surveyed, no connection of the bulb to the tubular portion of the complex could be demonstrated. Flocculent material was in both types of bulbs, yet only in connected bulbs was it evenly dispersed. Here its appearance closely resembled the cytoplasm of the head region proper. Because the flocculent material was clumped in the disconnected bulbs, areas devoid of this substance were especially electron translucent (figs. 19, 21). In sections of “disconnected” bulbs, the Sertoli endoplasmic reticulum was either not as extensive or was absent from its characteristic position facing the bulbs. It should be emphasized that although serial section of this type of bulb was not undertaken to conclusively demonstrate this discontinuity, extensive examination of many hundreds of this type of bulb never showed it to be connected to the spermatid head proper. Mid-Stage VII By mid-Stage VII very large vacuoles were seen in juxtaposition to the clustered bulbs (figs. 20-24). Within these vacuoles were two or more smaller, membrane-bound vesicles and a dense substance which filled in the gap between these vesicles (figs. 20-23). Lysosomes displaying dense material of a similar nature were numerous in the area of the vacuoles (figs. 20, 241, and in several instances the bounding membranes of the lysosome encircled or partially encircled bulbous endings (figs. 20-23). Upon closer examination of the vesicular structure contained within vacuoles, i t was found t h a t many displayed aggregated flocculent material of the type seen within the bulbous portions (disconnected bulbs) of the complex (figs. 20, 23). The space between the membranes of the vesicle and t h a t of the bounding membrane of the vacuole (in regions where they came into close apposition) was about 4.0 nm (figs. 21, 22). Based upon size, contents, appearance and morphological relationships, it was assumed that the structures within the phagocytic vacuoles (lysosomes containing bulbs) were bulbous endings which were freed from their connection to the spermatid tube and were in the process of undergoing dissolution. During mid-Stage VII the vesicles contained within the phagocytic vacuoles apFigs. 17-19 Early Stage VII. Fig. 17 Forming tubulobulbar complex at the dorsal convex aspect of a step 19 spermatid. A bristle-coated pit, seen at the depth of a Sertoli invagination, is associated with a short evagination of the spermatid plasma membrane. The spermatid plasma membrane “thins” down considerably (i.e., the glycocalylx is lost) in the region of the evagination. x 75,000. Fig. 18 Newly formed tubulobulbar complex at the ventral (concave) aspect of the spermatid. The tube remains constricted in both its proximal and distal portions; however, a central bulb has developed which is flanked by smooth endoplasmic reticulum of the Sertoli cell. A bristle-coated pit is present at the depth of the Sertoli recess. X 46,000. Fig. 19 Section of the Sertoli apical expansion showing the spermatid head and several clustered bulbs. Two types of bulbs are present; smaller bulbs with evenly distributed flocculent material, and larger ones with a patchy distribution of this same type of material. The smaller ones demonstrate association with Sertoli smooth endoplasmic reticulum and a connection with the tubular portion of the complex, whereas the larger ones have little or no association with this reticulum and do not demonstrate a connection with the tubular portion of the complex. X 38,800. TUBULOBULBAR COMPLEXES OF THE RAT 221 222 LONNIE D. RUSSELL peared smaller, developed irregular outlines, and thus were barely recognizable as vesicular structures. Several large phagocytic vacuoles of the type just described may be seen within each apical Sertoli expansion, although the contents of each may be in different stages of dissolution (fig. 20). During the period in which the large bulbous endings (those formed in early Stage VII) were being resorbed, attention was directed toward determining the influences of those events on the status of the remaining tubulobulbar elements in the concavity of the sickleshaped head. The following morphological observations were recorded: (1) tubulobulbar complexes were about as numerous, or more so, in mid-Stage VII as in early Stage VII; (2) the general arrangement of these structures within the apical Sertoli expansion remained unchanged from that of early Stage VII; (3) tubulobulbar complexes in an early phase of development were occasionally seen a t this time (fig. 23); (4) bulbous endings of tubulobulbar complexes were smaller (0.2-0.5 pm across) in mid-Stage VII and more elongated than those seen in early Stage VII (like that shown in fig. 25). Late Stage VII and early Stage VIII In late Stage VII and early Stage VIII the ectoplasmic specializations (filaments and more deeply placed endoplasmic reticulum) were displaced from their position facing the head of the spermatid (figs. 23, 24). Large phagocytic vacuoles and the smaller primary lysosomes were also a conspicuous feature in the apical Sertoli droplet in late Stage VII and early Stage VIII (figs. 23,241. The contents of some vacuoles, because of distortion of their internal membranes, were in an advanced stage of degradation whereas others appeared in the initial stages of resorption. Sections which reveal the tubular portion of the complex to best advantage showed that many of these tubular structures were also in the process of being resorbed. In cross sections, the spermatid tube was often absent, or when present, its plasma membrane appeared discontinuous (fig. 27). When sectioned longitudinally, the spermatid component of the tube was represented by stacked cylinders along the tube with “empty” areas seen between each cylindrical fragment (figs. 25, 26). Some fragmented tubes of the type just described could be traced to bulbous structures whereas others terminated blindly facing a Sertoli bristle-coated pit (fig. 26). With time, further regressive changes were observed in the tubular portions of the complexes, which led t o the eventual resorption of these structures. At the same time these regressive phenomena were noted, it was found that new tubulobulbar complexes were being formed. This was evidenced by bristle-coated pits in the depth of Sertoli invaginations and short opposing evaginations of the spermatid plasma membrane, all of which were found along the dorsal convex and lateral aspects of the spermatid (also seen in mid-Stage VIII; fig. 28, inset). When fully formed, the evaginated spermatid tube usually extended a variable distance to end blindly facing a Sertoli bristle-coated pit. A bulbous component was usually not part of this complex. Mid-Stage VIII The apical expansion in which the head of the late spermatid is embedded was gradually withdrawn (Russell and Clermont, ’76; Russell, ’77b). The mass of Sertoli cytoplasm occupying the concavity of the sickle-shaped head was first to be withdrawn leaving smaller processes of Sertoli cytoplasm in contact with the dorsal convex and the lateral surfaces of the head (figs. 28,291. Appropriate sections revealed that both intact and fragmented tubulobulbar complexes extended into Figs. 20-22 Mid Stage VII: ferrocyanide:osmium treated tissues. Fig. 20 Cross section of the spermatid head and the apical Sertoli expansion. Lysosomes and lipid droplets (li) and phagocytic vacuoles are seen in the region where bulbs are clustered. Evenly dispersed flocculent material lies within some of the bulbs; in others this material appears clumped. Only in the former does the Sertoli smooth endoplasmic reticulum face the bulb. One bulb (isolated arrow) appears to be engulfed (or surrounded) by a phagocytic vacuole. X 34,000. Fig. 21 Apical Sertoli expansion showing the bulbous portions of two tubulobulbar complexes. Flocculent material within these bulbs appears clumped. Note the absence of associated smooth endoplasmic reticulum. In most areas the Sertoli and spermatid membranes are very close together (about 4.0 nm), however, in one region, small vesicles and an electron dense lysosome-like material lie between the spermatid and Sertoli plasma membranes. X 46,000. Fig. 22 A small bulb is shown a t the upper right portion of this figure within a n apical Sertoli expansion. It displays evenly dispersed flocculent material and facing Sertoli endoplasmic reticulum. At the lower left is a large phagocytic vacuole containing numerous internally p i tioned bulbs. These bulbs display aggregates of flocculent material and are separated from the Sertoli plasma membrane by a 4.0 nm space (arrow). A dense substance and small vesicles (v) fills the gap between phagocytised bulbs and the Sertoli plasma membrane. x 67,000. TUBULOBULBAR COMPLEXES OF THE RAT 223 224 LONNIE D. RUSSELL these Sertoli processes. Tubulobulbar complexes were in evidence through mid-Stage VIII and up until near the time of sperm release; however, as the sperm release zone was approached, these complexes generally appeared fragmented (fig. 29). Late Stage VIII In longitudinal sections of seminiferous tubules during Stage VIII it has been shown that all but a few sperm are released a t a specific point along the tubule (Perey et al., '61; Russell, '77a). After release, residual bodies line the surface of the seminiferous epithelium. Of the few remaining spermatids encountered, some were abnormal in appearance (fig. 30). In cells of this type that were malformed, the defect generally involved a malformation in the acrosomal region. Especially interesting was the relationship of these abnormally shaped cells to the Sertoli cell. It was similar to that noted for mid and late Stage VII, i.e.: (1)the Sertoli apical expansion circumscribed the head of the spermatid, (2) ectoplasmic specializations were present facing the spermatid head, and (3) intact tubulobulbar complexes were in evidence (fig. 30, inset). Acid phosphatase cytochemistry In late Stage VII and early Stage VIII acid phosphatase activity could be demonstrated within saccules of the Golgi of all germ cells as well as within boundaries of lysosomes throughout the testis, including the large, clear vacuolar structures of the cytoplasmic lobe.' As evidenced by an electron dense lead precipitate, the tubular portions of many complexes were found to be reactive (figs. 32, 33) as were nearby phagocytic vacuoles (fig. 31). Control tissues were without reactivity. DISCUSSION The tubulobulbar complex is observed in two widely separated areas of the seminiferous epithelium (Russell, '75; Russell and Clermont, '76; Russell, '79). The present report details further observations on tubulobulbar complexes formed between Sertoli cells and late spermatids. Structure and relationships of the tubulobulbar complex me freeze-cleave technique was employed to d&ermine if tJlbulobulbar plasma membranes were in any way different from those plasma membranes of the spermatid head proper or of the Sertoli cell. Frozen-cleaved Sertoli membranes of the tubulobulbar complex were shown to possess few membrane associated particles. It was thought that the E face of the Sertoli plasma membrane around the spermatid tube might reveal membrane particles associated with attachment sites of subsurface filaments or associated with junctional proteins. These features, however, could not be demonstrated with the freeze-cleave technique. Most fracture faces identified were those of the Sertoli cell, although there were sufficient fractures of spermatid plasma membranes to show that the internal aspects of these membranes were likewise nearly particle-free. In thin-sectioned material the extracellular aspects of the membranes of the complex were examined for evidence of a cell coat. Although a substantial glycocalyx was noted on the spermatid plasma membrane of the head proper, this feature was lost as the membrane became continuous with that of the spermatid tube of the tubulobulbar complex. Nicander ('67) has also observed a "thinning out" of the spermatid plasma membrane of the head as it became the membrane of the spermatid tube. The Sertoli plasma membrane of the tubulobulbar complex also lacked a visible glycocalyx. Thus, both Sertoli and spermatid plasma membranes of the complex appeared quite thin, measuring about 7.5 nm in thickness; and each was deficient of internal mem' The term cytoplasmic lobe ("lobe protoplasmique"; Regaud, '01) is used to designate that excess cytoplasm of the spermatid which extends from this cell toward the base of the tubule. Once the cytoplasmic lobe is freed from the spermatid it is termed the resrdual body. Fig. 23 Apical Sertoli expansion showing the head of the step 19 spermatid in mid-Stage VII and portions of several tubulobulbar complexes. A large phagocytic vesicle containing two bulbous endings is seen at the bottom of the micrograph. In this same micrograph a developing tubulobulbar complex is present. All bulbs containing aggregations of dense material. x 42,000. Fig. 24 Several spermatids circumscribed by apical Sertoli expansions are shown in this late Stage VII tubule. The bold arrow at the top of the figure points to the basal aspect of the same Sertoli cells which reach to the lumen to surround these spermatids. Both tubular and bulbous portions of the complex are indicated as well as lysosomes and a phagocytic vacuole. Sertoli ectoplasmic specializations have shifted from a position facing the heads of the late swrmatids to one considerablv closer to the base of the tubile. Here they are seen alongthe apical Sertoli stalk, either at its free edge or, in some cases, internalized within the cell. Tissue treated with ferrocyanide:osmium. x 11,000. TUBULOBULBAR COMPLEXES OF THE RAT 225 226 LONNIE D. RUSSELL brane associated particles as well as an external cell coat. An especially narrow intercellular space (4.0 nm) was recorded between the two plasma membranes forming the tubulobulbar complex, which, at first, was thought to be indicative of some type of junctional relationship between the two cells. The opposite point of view was taken after the freeze-cleave technique showed no significant number or particular arrangement of membrane-associated particles. Lacking a glycocalyx, these plasma membranes may be allowed t o reside much closer to one another, t o a degree not seen in other areas where the same two cells are adjoining. Since membranes of both the tube and the bulb exhibit this close spacing, it can be assumed that Sertoli filaments. which are only present around the tube, are not responsible for drawing the two cells this close together. The type of subsurface filamentous condensation around the tube appears similar in appearance t o the contractile ring of dividing cells. Further studies are needed t o determine if these filaments are actin or actin-like in nature. Formation and resorption of tubulobulbar complexes To minimize possible error in interpretation of numerous sequential events, it was found to be advantageous t o subdivide (based on morphology) known stages of the cycle into several substages (MATERIALS AND METHODS). During these time periods, a likely series of events can be constructed from micrographs which depict the formation and dissolution of tubulobulbar complexes. The sequence given in figure 34 is based on the observations presented herein. The smallest profiles of tubulobulbar complexes, observed in early Stage VII, consist of short tubular evaginations of the spermatid which are found within corresponding Sertoli invaginations (fig. 34B). A bristle-coated pit is always present in the depth of the recess formed by the Sertoli cell. Bristle-coated pits were not uncommon along the plasma membrane which faces the head of the spermatid (fig. 34A) and i t is, therefore, tempting to suggest that since these are the first structures visible that they are responsible for initiating the formation of this complex. Sertoli cells are well equipped, by virture of possessing an abundance of microfilaments and micro- tubules, t o accomplish the shape changes necessary in the development of tubulobulbar complexes, whereas the spermatid head visiibly lacks these cytoplasmic organelles. Christensen ('76) has reported that small processes of a Leydig cell may protrude for a short distance into neighboring macrophages. Although the structures described by Christensen show little resemblance to tubulobulbar complexes, the two cell-to-cell relationships share one feature in common. A bristle-coated pit is always seen in the depth of the recess formed by the invaginated cell. Available evidence indicates that bristle-coated pits, of the type seen in this study, are important in the uptake of materials from outside the cell (Roth and Porter, '64; Lagunoff and Curran, '72). To this author's knowledge, they have not been shown to be involved in the uptake of portions of another cell. As the tube elongates (figs. 34C,D) and develops a bulbous expansion (figs. 34C,D), appropriate sections show that the bulb develops in the mid-region of the complex. Thus, the distal portion of the complex remains narrow and faces a bristle-coated pit in the deepest portion of the Sertoli recess. This observation necessitates a slight modification of our conFigs. 25-27 Late Stage VII or Early Stage VIII; secondarily treated with a ferrocyanide:osmium mixture. Fig. 25 Apical Sertoli expansion showing a tubulobulbar complex in longitudinal section and a nearby bulbous component. The spermatid tube is fragmented and has separated from the bulb. X 25,000. Fig. 26 Longitudinal sections of tulbulobulbar complexes showing that these structures lack a bulbous component. The spermatid tube is discontinuous in many areas and is represented by stacked cylindrical masses within the invaginated Sertoli cell. At the depth of the recess, a coated pit opposes the terminal endings of the spermatid tube. x 14,000. Fig. 27 This section through the apical Sertoli expansion shows several tubes in cross sectional profile. The spermatid plasma membrane which forms the tubular portion of the complex is either absent or poorly represented. Sertoli filaments remain a conspicuous structure in a position surrounding the Sertoli plasma membrane. X 45,000. Fig. 28 Survey micrograph showing the relationship of the late spermatids to the Sertoli cell in mid-Stage VIII. The direction of the basal aspect of the Sertoli cell, since only the apical processes of these cells are seen in this micrograph, is indicated by the bold arrow. These apical processes extend toward the lumen to make contact with the spermatid a t its dorsal convex and lateral surfaces. Numerous tubulobulbar complexes are seen to extend from the spermatid into these processes. The excess spermatid cytoplasm or cytoplasmic lobes are also indicated (cl). The inset shows an enlargement of one of these spermatids and the profile of a developing tubulobulbar complex. Tissue post-treated with osmium. X 5,200. 227 TUBULOBULBAR COMPLEXES OF THE RAT L 228 LONNIE D. RUSSELL cept of the general shape of the tubulobulbar complex as shown in figure 34D. The data strongly suggest t h a t more than one generation of tubulobulbar complexes form during late spermiogenesis. Observations to support this suggestion are as follows: (1) Many bulbous components of the complex undergo dissolution in mid-Stage VII (figs. 34E,F), yet as many intact tubulobulbar complexes (displaying both tube and bulb) remain; (2) Tubulobulbar complexes of early Stage VII primarily emanate from the ventral aspect of the spermatid whereas those in Stage VIII protrude from dorsal convex and lateral aspects of the cell (fig. 34G); (3) Bulbous components of the complex in mid-Stage VII are considerably smaller than those initially formed in early Stage VII (figs. 34E-GI; (4) Newly formed tubulobulbar complexes are seen throughout Stage VII and Stage VIII (fig. 34F). The preceding observations are consistent with what might be considered aprocess of active formation and resorption o f tubulobulbar complexes in the period prior to sperm release. Assuming that more than one generation of tubulobulbar complexes is formed, it is soon realized that a substantial portion of the spermatid membrane and cytoplasm is resorbed in the 4-day period prior to sperm release. Considering the resorption of just one large complex, the tubular portion measuring 4.0 p m in length and the bulbous portion measuring 2.0 p m across, the loss of surface membrane would be approximately 12.0 p m ' and the volume of cytoplasm resorbed would be approximately 4.0 pm3. Up to 24 tubulobulbar complexes associated with one spermatid may be detected in a single section (Russell and Clermont, '76). Acid phosphatase cytochemistry The results from acid phosphatase cytochemistry confirm that it is the phagocytic activity of the Sertoli cell which is important i n resorbtion of tubulobulbar complexes. Acid phosphatase is a well known hydrolytic enzyme of lysosomes, which, together with other lysosomal enzymes, acts to digest material taken up by cells. Lysosomes are present within the Sertoli cell, in and around the clustered bulbous portions of the complex, and become more prevalent as time passes. Both the phagocytic vacuoles and the tubular portions of the tubulobulbar complex displayed dense lead deposits which demonstrate that acid phosphatase activity is present a t these sites. The precise manner in which enzymes are transferred from within the Sertoli cell into the tubular and bulbous portions of the complex remains to be elucidated; however, the nearly particle-free nature of the membrane of the bulb may facilitate fusion between bulb and lysosome as has been shown for other membrane systems (Lucy, '70; Ahkong et al., ('75). Tubulobulbar complexes and spermiation Relatively little information is available about the spermiation process. Most investigators, including this author, are in general agreement with the work of Fawcett and Phillips ('69) which indicates that the Sertoli cell, by actively changing its configuration, influences the release of late spermatids. It is difficult to envision that the spermatid might be the sole active agent in this process, for during spermiogenesis, the spermatid had been transformed into a highly specialized gamete, which, in its near final form, hardly seems capable of engineering its own release. Fig. 29 Figure shows the relationship of the head of t h e late spermatid to the apical Sertoli cell process. The micrograph was taken from a specific area of a longitudinal section of a seminiferous tubule in which t h e sperm release zone was about 0.2 millimeter further along t h e tubule. Apical Sertoli processes have withdrawn from t h e ventral concave aspect of t h e spermatid and are related only to t h e dorsal convex and lateral aspects of the spermatid. Tubulohulbar complexes are seen extending from the dorsal aspect of t h e spermatid into this apical process. Microtubules (mt) are numerous within this apical Sertoli process and extend along its long axis. Post-treated with osmium. X 12,000. Fig. 30 Survey micrograph of late Stage VIII showing the surface of the seminiferous epithelium. Residual bodies (rb) are essentially the most superficial structures since the apical Sertoli processes have been withdrawn toward the base of t h e tubule. All spermatids have been released except the two shown a t the upper left of the figure. In one of these (see inset) the acrosome displays a bizarre configuration. Here, a n apical Sertoli expansion is present circumscribing the head. Also, ectoplasmic specializations of the Sertoli cell face the head and the presence of intact tubulobulbar complexes is indicated by arrows. X 4,300; x 20,000. Figs. 31-33 Acid phosphatase preparations of l a t e Stage VII and early Stage VIII. Fig. 31 Dense reaction product is present within a large phagocytic vacuole, yet this precipitate lies external to phagocytosed bulbs. x 27,000. Fig. 32 Longitudinal section of a n apical Sertoli expansion showing reaction product within cross-sectional profiles of t h e tubulobulbar complex (bold arrows). Some tubes show little or no reaction product (small arrows). x 12,000. Fig. 33 This longitudinal section of t h e tubular portion of a tubulobulbar complex shows a heavy disposition of reaction product. Sertoli filaments are seen a t the periphery of the reaction product. x 39,000. TUBULOBULBAR COMPLEXES OF THE RAT 229 230 LONNIE D. RUSSELL TUBULOBULBAR COMPLEXES OF THE RAT Recent reports have indicated that ectoplasmic specializations are important in the spermiation process. Ross and colleagues have shown experimentally that the subsurface bundles of filaments and associated endoplasmic reticulum, which comprise the ectoplasmic specialization, are sites associated with cell adhesion between the spermatid and the Sertoli cell (Ross, '76; Romrell and Ross, '78). Others have suggested that the ectoplasmic specialization maintains a grasp on the head of the spermatid (Toyama, '76; Russell, '77b) andlor acts as a motile apparatus which may transport the late spermatid (Russell, '77b; Gravis, '78). All investigators agree that this surface specialization is important in preventFigs. 34A-H Diagram summarizing the steps in the formation and dissolution of tubulobulbar complexes in Stages VII and VIII. This drawing depicts a likely series of events which is based on numerous electron micrographs from these stages. The Sertoli cytoplasm is shown in yellow. A Cross section of a spermatid head showing the nucleus (n), perinuclear cytoplasm (pc), acrosome (a), Sertoli cell apical expansion ( 8 ) and Sertoli ectoplasmic specialization (es). A bristle-coated pit is seen in the Sertoli plasma membrane facing the head of the spermatid (arrow). Early Stage VII. B An evagination of the spermatid is seen within a corresponding recess of the Sertoli cell. The spermatid tube thus formed faces a bristle-coated pit. Filaments are seen within the area of the Sertoli cytoplasm which faces this spermatid projection. early Stage VII. C The spermatid tube elongates, and its midportion dilates to form the bulbous portion of the complex. Sertoli filaments are present, facing both the proximal and distal narrowings forming the tube. Early Stage VII. D Further elongation of the proximal spermatid tube takes place such that the complex now extends 3.0-5.0 p m into the apical Sertoli expansion. A short distal spermatid tube is present which ends facing a Sertoli bristle-coated pit. Lysosomes are shown in the region of the clustered bulbs. Early Stage VII. E In early and mid-Stage VII many bulbs appear to lose their connection to the spermatid proper. The dense material within disconnected bulbs appears clumped. F In mid-Stage VII new tubulobulbar complexes form. Meanwhile large vacuoles containing phagocytized bulbs are present in the vicinity of intact bulbs. G In late Stage VII and Early Stage VIII, ectoplasmic specializations facing the spermatid head become displaced and are then seen mainly along the peripheral aspect of the Sertoli cell. Fragmentation of the spermatid tube. takes place. The bulbous portions of many tubulobulbar complexes are seen facing a Sertoli bristlecoated pit. H By mid-Stage VIII the apical Sertoli expansion faces only the dorsal convex andlor lateral aspects of the spermatid head. Tubulobulbar complexes (without lulbs) form and extend into this Sertoli cell process. Prior t ) sperm release these complexes also dissociate leaving 1.3 trace of tubulobulbar complexes on the mature spermatozoan. 231 ing premature sloughing of germ cells, and that the loss of the relationship of the germ cells with this surface specialization of the Sertoli cell facilitates sperm release. The present study has shown that tubulobulbar complexes are present long after Sertoli ectoplasmic specializations have left their position facing the spermatid (fig. 34g). From the configuration of these complexes it appears that they must make some contribution to the stability of the relationship between the spermatid head and the Sertoli cell. This relationship persists until near the time of release when their dissolution from the dorsal region of the head takes place. The relative importance of tubulobulbar complexes as anchoring devices when compared with other possible stabilizing factors has yet to be ascertained. Beyond the zone in which most spermatids had been released (in longitudinal sections of Stage VIII) was an area in which residual bodies, and on occasion a few abnormally shaped sperm, were seen near or bordering the tubular lumen. The relationship between the Sertoli cell and these late spermatids had not progressed as was described for the normally shaped spermatids but had remained a t the same developmental stage as seen in midStage VII. In evidence were: (1) The apical Sertoli expansion around the head; (2) opposing Sertoli ectoplasmic specializations and (3) intact tubulobulbar complexes. Since these few sperm were not released, as were others of the same generation, it might be assumed that one or more of these features was important in preventing this action. The persistence of a type of relationship between the two cells like that just described may be a means by which abnormal spermatids are retained and prevented from traversing the male excurrent duct system. ACKNOWLEDGMENTS The generosity of Doctor Donald Caspary in providing an electron microscope facility a t the Springfield Campus is acknowledged. Many thanks are due Doctors David Smith and Marilyn Cayer and the Papanicolau Cancer Research Institute in Miami, Florida, for providing help and allowing the use of equipment for the freeze-cleave studies. The help of Mr. J. Malone and Mr. J. Ostenburg in the preparation of the manuscript is also appreciated. This work was supported by a Grant from the Institute of Child Health and Human Development (NIH-11823). 232 LONNIE D RUSSELL LITERATURE CITED Ahkong, Q. F., D. Fisher, W. Tampion and J. A.Lucy 1975 Mechanism of cell fusion. Nature (London),253: 193-195. Barka, T., and P. J. Anderson 1963 In: Histochemistry: Theory, Practice and Bibliography. Harper and Row, Publishers, Inc., New York, p. 240. Branton, D., S. Bullivant, N. B. Gilula, M. J. Karnovsky, H. Moor, K. Muhlethaler, H. Northcote, L. Packer, B. Satir. P. Satir, U. Speth, L. A. Staehelin, R. L. Steere and R. S. Weinstein 1975 Freeze etching nomenclature. Science. 190: 54-56. Christensen, A. K. 1976 Specific contacts between Leydig cells and macrophages in t h e rat testis. Anat. Rec., 184: 377 (Abstract). Clermont, Y.,C. P. Leblond and B. Messier 1959 Duree du cycle de l’epithelium seminal du r a t . Arch. Anat. Microscop. Morph. Exptl., 48: 36-55. Fawcett, D. W.. and D. M. Phillips 1969 Observations on the release of spermatozoa and on changes in the head during passage through the epididymis. J. Reprod. Fertil. (Suppl.), 6: 405-418. Gravis, C. J. 1978 A scanning electron microscopic study of the Sertoli cell and spermiation in t h e Syrian Hamster. Am. J. Anat., 151: 21-38. Karnovsky, M. J. 1971 Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. J. Cell Biol., Abstract No. 284. Lagunoff, D., and D. E. Curran 1972 Role of bristle-coated vesicles in the uptake of ferritin by rat macrophages. Exptl. Cell Res., 75: 337-346. Leblond, C. P., and Y. Clermont 1952 Definition of the stages of the cycle of t h e seminiferous epithelium in t h e rat. Ann. N. Y.Acad. Sci., 55: 548-573. Lucy, J. A. 1970 The fusion of biological membranes. Nature (London). 227: 815-817. Nicander, L. 1967 An electron microscopical study of cell contacts in seminiferous tubules of some mammals. 2. Zellforsch., 83: 375-397. Perey. B., Y. Clermont and C. P. Leblond 1961 The wave of t h e seminiferous epithelium of the rat. Am. J. Anat., 108: 47-78. Regaud, C. 1901 Etudes sur la structure des tubes seminiferes e t sur la spermatogenese chez les mammiferes. Arch. Anat. Microscop., 4: 101-156; 231-380. Romrell, L. J., and M. H. Ross 1978 Sertoli cell-germ cell junctional specializations in isolated rat testicular cells. Anat. Rec., 190: 523 Abstract. Ross, M. H. 1976 The Sertoli cell junctional specialization during spermiogenesis and a t spermiation. Anat. Rec., 186: 79-104. Roth. T. F., and K. R. Porter 1964 Yolk protein uptake in the oocyte of the mosquite Aedes aegypt L. J. Cell Biol., 20: 313-332. Russell, L. 1977a Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am. J. Anat., 148: 313-328. 197% Observations on rat Sertoli ectoplasmic (“junctional”) specialization in their association with germ cells in the rat testis. Tissue and Cell, 9: 475-498. 1979 Observations on the inter,relationships of Sertoli cells a t t h e level of t h e blood-testis barrier: Evidence for formation and resorbtion o f Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle. Am. J. Anat., in press. Russell, L., and S. Burguet 1977 Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture. Tissue and Cell, 9: 751-766. Russell, L., and Y. Clermont 1976 Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules. Anat. Rec., 185: 259-278. Russell, L. D. 1975 A new type of spermatid-Sertoli cell and Sertoli-Sertoli cell connection in the rat testis. Anat. Rec., 181: 469-470 (Abstract). Toyama, Y. 1976 Actin-like filaments in t h e Sertoli cell junctional specializations in the swine and mouse testis. Anat. Rec., 186: 477-491. Vitale, R., D. W. Fawcett and M. Dym 1973 The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment. Anat. Rec., 176: 333-344. Note a d d e d in proof.. The author is grateful to Dan Friend for recently pointing out the morphological similarities in the relationship of the Sertoli cell and late spermatid (as demonstrated in this study) to that of the rod cells of the retina and the covering pigment epithelium. Pigment cells of the retina cap the rod outer segments and are involved in phagocytosis of detached disks (Young, R. W., ’76, Invest. Ophth., 15: 700725). In this manner a portion of a living cell is degraded by a neighboring cell. The Sertoli cell performs a similar task.