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Effect of the microtubule disrupting agents, colchicine and vinblastine, on seminiferous tubule structure in the rat
TISSUE & CELL 1981 13 (2) 349-367 0 1981 Longman Group Ltd
LONNIE D. RUSSELL, JAMES P. MALONE and DANIEL S. MacCURDY
EFFECT OF THE MICROTUBULE DISRUPTING AGENTS, COLCHICINE AND VINBLASTINE, ON SEMINIFEROUS TUBULE STRUCTURE IN THE RAT ABSTRACT, Injections of colchicine or vinblastine were given intratesticularly and rats sacrificed 6 and 12 hr later. Colchjcine and vinblastine produced identical morphological patterns of response in the seminiferous tubules resulting in arrest of germcell mitoses and meioses and a rapid depletion of the microtubules normally found within the Sertoli cell. Sloughing of cells into the lumen of seminiferous tubules was the most prominent feature noted. Germ cells and portions of the apical Sertoli cells were frequently sloughed together where they remained in close association. Usually germ cells and associated Sertoli cell fragments were cleaved from the wall of the seminiferous tubule at a level between dissimilar generations of germ cells, e.g. between spermatocytes and spermatids. This selective sloughing probably occurred as the result of the support normally provided by intercellular bridges which link clones of like germ cell types. Sequential steps in the process leading to sioughing of Sertoli-germ cell associations could be inferred from observations made in plastic 1 pm sections. Cell sloughing at 12 hr post-injection was generally more extensive. It was frequently noted that germ cells and the apical portions of Sertoli celts had been extruded to the level of the most adluminal tight junctions forming the blood-testis barrier. It was concluded that disruption of Sertoli microtubules was responsible for sloughing of Sertoli fragments and associated germ cells, and that the cytoskeletal support of the Sertoli cell was, at least in part, dependent upon the integrity of Sertoli microtubules. The Sertoli cell could not round-up after loss of its cytoskeletal support, due to the numerous attachment devices known to link it with various apically positioned germ cells. Thus, the cell was severed at some point along its delicate apical processes, as the consequence of forces produced by the ‘rounding-up’ process. Long-term sacrifice after vinblastine or colchicine treatment allowed the Sertoli cells to regain microtubules and long processes but not their typical configuration. Spermatogenesis remained severely impaired.
Introduction
Until recently, its three-dimensional configuration had not been realized (Wong and Russell, 1981; Russell and Wong, 1981). These studies employed reconstruction techniques to make a Sertoli cell model. The Stage V reconstructed Sertoli cell extended from the base of the seminiferous tubule to the tubular lumen and, although irregularly shaped, could generally be regarded as a columnar cell. The reconstructed model represented only one of two major architectural forms of the Sertoli cell, since the Sertoli cell is thought to change its basic configuration in
OF all the cells in the body, the Sertoli cell is one of the most complex, both from a structural and a functional standpoint (Fawcett, 1975; Russell, 1980). The fine details of its in vivo shape have long been hypothesized, based on evidence provided by studies employing random thin sections. Department of Physiology and School of Medicine, Southern Illinois University, Carbondale, IL 62901. Received 6 November 1980. Revised 7 February 198 I. 349
350 stages (VII-VIII) of the spermatogenic cycle which prepare the sperm for its eventual release (Fawcett and Phillips, 1969; Russell and Clermont, 1976; Gravis 1978). Throughout the cycle, the Sertoli cell closely conforms to the outlines of the germinal cells and, in certain regions, to other Sertoli cells. By virtue of its configurational relationships to nearby germ cells, it has long been hypothesized to be important in the development and release of these cells (Fawcett, 1975; Russell, 1980). Microtubules have generally been considered as important structures in the maintenance of cell architecture (asymmetrical cell shape) such as that displayed by the Sertoli cell. They do so by acting as semi-rigid skeletal elements supporting (Byers and Porter, 1964; Fawcett and Witebski, 1964; Tilney and Porter, 1965; and many others) although they may act in concert with the system of microfilaments or other support elements (Pollard, 1980; Griffith, 1980; Wolosewick and Porter, 1976). A large body of evidence showing that microtubules are important in the attainment and/or maintenance of cell shape is derived from studies in which the destruction of microtubules has led to a change in cell shape and/or studies which have demonstrated a preferential orientation of microtubules in the long axis of a cell or cell process (Dustin, 1978; Ham, 1974; Fawcett, 1975). Christensen (1965) first described microtubules within the Sertoli cell of the guineapig as being abundant and extending radially within the cell (from the base to the lumen). This particular orientation of microtubules in the long axis of the Sertoli cell has been confirmed by Fawcett (1975). Christensen felt that microtubules ‘may support the tenuous cytoplasmic extensions of the Sertoli cells, and produce the various movements that spermatids undergo in the epithelium over the course of spermatogenesis. These concepts have never been tested experimentally, although we note the work of Wolosewick and Bryan (1977) and Handel (1979) who studied the effects of microtubule disrupting agents on spermiogenesis in the mouse, and Rattner (1970) who studied the effects of colchicine on In these spermiogenesis in guinea-pigs. studies, effects on Sertoli cells were not
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specifically examined; however, after treating mice with colchicine, Handel (1979) noted vacuolization of the Sertoli cell and apical Sertoli processes bulging into the tubular lumen. Wolosewick and Bryan (1977) and Rattner (1970) remarked on the rapid disappearances of Sertoli cell microtubules subsequent to treatment with microtubule destabilizing drugs. A very recent report (Aoki, 1980) describes the effects of microtubular agents injected into the lumina of the seminiferous tubules. Early changes including a fragmentation and retraction of Sertoli cell processes were suggested to be responsible for premature sperm release. In the present study, the microtubular disrupting agents, vinblastine and colchicine, were used for the primary purpose of observing the short-term changes in the Sertoli cell with special attention paid to changes related to its configuration. When administered appropriately, these drugs caused marked alterations in Sertoli-cell architecture which led to massive disruption of spermatogenesis. Materials and Methods Injections Twelve adult, male, Sprague-Dawley rats weighing 250-300 g were immobilized and one testis was injected, using a 32 gauge needle, with either colchicine (5 x 10m5 mg in 0.5 ml normal saline) or vinblastine (2.5 x 10m5 mg in 0.5 ml normal saline). Six control rats were given saline alone (0.5 ml). Prior to injection, the scrotal skin was shaved and cleaned with a generous amount of 70% ethanol. Under strong light, the tortuous aspect of the testicular artery was revealed. The juncture of the caudal third with the middle third of the testis was injected without rupturing blood vessels. Injections were made into the central area of the testis. Tissue preparation One-half of the animals in treatment and control groups were sacrificed six hr postinjection with the other half at 12 hr; and the tissue prepared for light and electron microscopic observation. To do this, animals were anesthesized with Nembutal and the testis perfused-fixed for 45 min with 5% gluteraldehyde in 0.2 M caccodylate buffer (ph 7.4) by a retrograde abdominal aorta
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method (Vitale et al., 1973). Subsequently, testis tissue within 3 mm of the injection site (as roughly estimated) and at the proximal pole of the testis were diced into small cubes and immersed in the same fixative solution for 30 min. Post-fixation was performed for 1 hr employing an osmium: ferrocyanide mixture (Russell and Burguet, 1977). After a brief wash in buffer, tissues were subsequently dehydrated, infiltrated and embedded in Araldite. Blocks were oriented such that round or nearly round (cross-sections) and longitudinal sections of tubules would be produced upon sectioning at 1 pm with an MT-1 Porter-Blum ultramicrotome. Thin sections of silver and silver-gold interference colors were obtained with a Sorvall MT-2 ultramicrotome and examined on a Philips 201 electron microscope. Long-term
experiments
For long-term experiments, three rats were injected with either vinblastine or colchicine at the specific dosages and volume of vehicle described above. Three control rats were injected with saline only. At twelve hours post-injection, the right testis was surgically removed and processed for light and electron microscopic observation. Perfusion fixation was accomplished with a 26-gauge needle inserted into the testicular artery. All rats were sacrificed sixty days later, whereupon the left testis was removed and perfusedfixed through the testicular artery. Tissues were prepared in a manner similar to that described above for the short-term experiments. Results Safine controk Animals injected with saline showed normal testicular structure at each of the 6 and 12 hr sacrifice intervals selected (Fig. 1). There was no evidence of hemorrhage in the intertubular spaces or of disruption or injury to the seminiferous tubules. Germ cells remained in their typical position (Russell, 1980) within the seminiferous epithelium. Microtubules were distributed throughout the Sertoli cell, but were scarce in the region lateral to and below the Sertoli nucleus (near the limiting membrane). In the supranuclear region, microtubules were more numerous and were oriented radially (in the long axis of the Sertoli cell), as indicated by
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Christensen (1965) and Fawcett (1975). In all stages, except VII and VIII (classification of Leblond and Clermont, 1952), microtubules coursed alongside elongating and elongate spermatids which were positioned in deep recesses within the Sertoli cell. Microtubules were also seen in close relationship to cytoplasmic vesicles (Fawcett et al., 1971) and Sertoli ectoplasmic specializations (Russell, 1977b). In Stages VII and VIII, they extended within the ‘trunk’ of the Sertoli cell (see Elftman’s (1963) comparison of the form of a Sertoli cell to that of a tree) and into the small processes which SUJrounded the heads of late spermatids (Russell, 1979a; Russell and Malone, 1980). When the trunk region of the Sertoli ceil was CJOSSsectioned (sections obtained from longitudinally oriented tubules within tissue blocks), this thickened portion of the cell appeared as shown in Fig. 14. It displayed numerous microtubules, also cut in CJOSSsection, which traveled in the long axis of the cell. Mitotic and meiotic divisions occurred in a typical and predictable pattern as has been described from numerous kinetic studies. A few mitoses of spermatogonia were seen at the periphery of a seminiferous tubule in which divisions were known to OCCUJ. In Stage XIV, primary spermatocytes are known to undergo two rapid successive cytokineses (Russell and Frank, 1978). This cell division pattern was also observed in saline injected animals (Fig. 5). Treated animals Animals injected with either colchicine or vinblastine appeared to show identical responses. Consequently, the following description applies to testes treated with either agent. Six hours post-injection. Tissues within 3 mm of the injection site showed an identical response, both in terms of the pattern of response and the magnitude of response, to those taken more distally. Mitotic (Fig. 4) and meiotic (Figs. 6, 7) arrest was a common feature. Arrest of spermatogonial divisions was frequently seen (Fig. 4) and the arrested cells usually ringed the entire tubule. Numerous spermatocytes arrested at meiosis I and II were present in Stage XIV and I tubules. Many of these appeared degenera-
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tive and showed a marked basophillia with distortion and clumping of nuclear cytoplasmic components (Fig. 6). Spermatids undergoing early phases of elongation in Stages VIII, IX and X were apparently unaffected by the treatments. Microtubules were present and appeared to form and/or maintain a more or less typical manchette. The most conspicuously abnormal feature within the seminiferous tubules was the sloughing of germinal and non-germinal elements. Some tubules showed no evidence of cell sloughing (Fig. 2); but in most, this process took place to a variable degree (Figs. 2, 7, 8, 10, 12). Not only were germ cells shed, but the apical portions of Sertoli cells attached or related to many of these germ cells were broken away from their parent cells. Evidence for this process came from both light and electron microscopy, where it was observed that whole segments of the seminiferous epithelium appeared to be sloughed. This appeared to occur by a mechanism which involved cleavage of the Sertoli cell at some point above its nucleus (apical aspect). These sloughed Sertoli cellgerm cell associations were then freed into the tubular lumen (Figs. 2, 3, 7, 8, 10, 12, 16-l 8). Sloughed Sertoli cell fragments within the tubular lumen were frequently observed in the form of large rounded bodies indicating that the previously columnar aspect of the apical Sertoli cell had rounded-up (Figs. 16-18) during and/or after the process
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of being sloughed. Elongated spermatids were frequently observed within recesses of these rounded Sertoli profiles (Fig. 17). Germ cells undergoing the elongation process were arranged in ‘rosettes’ around a centrally positioned round Sertoli cell fragment (Figs. 16, 18). Sertoli fragments often appeared degenerate, showing ruptured membranes and some loss of cytoplasmic matrix (Fig. 17). Areas where breaks might have occurred between the parent Sertoli cell and the Sertoli fragment were difficult to identify; however, observations represented by Fig. 13 (12 hr post-injection rat) suggested recent separation of apical Sertoli processes. The apical processes in affected tubules were typically seen as tenuous extensions between the round germ cells which remained to line the tubular lumen (Fig. 12). Occasionally, Sertoli processes reached the lumen; but, large ‘trunks’ of Sertoli cells which had presumably undergone apical separation, appeared to have retracted except in regions where the epithelium was severely depleted. Sertoli cell fragments could be identified with the electron microscope by using characteristic ultrastructural features such as the presence of tubulobulbar complexes (Russell and Clermont, 1976; Figs. 12, 17), ectoplasmic specializations (Flickinger and Fawcett, 1967; Russell, 1977b; Figs. 17, 18), and the appearance of the mitochondria (Fig. 12). Once identified at the electron microscope level, large Sertoli fragments easily identified in 1 pm
Figs. 1-3. Light micrographs showing several cross-sectional profiles of seminiferous tubules, all of which were processed from an area near the site of injection. x 200. Fig. 1.This 6 hr, saline-injected control demonstrates a full complement of germinal cells and a typical association of cells forming the wall of the seminiferous epithelium. No cellular elements have been sloughed into the tubular lumen, and no hemorrhage is indicated in the intertubular spaces. Fig. 2. This colchicine-injected, 6 hr sacrificed animal shows regions of focal sloughing (arrows) and the presence of cellular debris in the lumina of some seminiferous tubules. Step 8 spermatids (arrowheads) are present in a Stage XIII tubule. Fig. 3. This colchicine-injected, 12 hr sacrificed animal shows massive sloughing of cellular elements as evidenced by depletion of cells from the seminiferous epithelium and the presence of loose cells within the tubular lumina. One tubule (at bottom center) shows sloughing of cellular elements, up to the region near the limiting membrane, leaving only one layer of germinal cells present (arrows).
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Figs. 4-6. Light micrographs of mitotic and meiotic cells, all of which were taken from tissues near the site of injection which have been sacrificed 6 hr post-injection. Fig. 4. This Stage VI seminiferous tubule, from a vinblastine-treated rat, was sectioned in a region where the tubule makes a bend. It shows several arrested spermatogonia in the process of dividing to form preleptolene spermatocytes (arrows). x 1250. Fig. 5. This Stage XIV (on the right) tubule from a saline-injected shows the typical appearance of dividing meiosis 11 cells. x 1100.
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Fig. 6. This Stage XIV tubule from a colchicine-injected testis shows the appearance of dividing meiotic cells, some of which appear atypical (arrows), others of which demonstrate astrong basophilia and are degenerating (arrowheads). Sloughing of some late spermatids (step 14) has occurred. x 1300. Figs. 771 I. Light micrographs of seminiferous tubules near the site of injection which have undergone various degrees of sloughing of germinal and Sertoli elements. Except for Fig. 9, which was taken from tissue prepared 12 hr post-injection, all photographs are from 6 hr post-injection animals. Fig. 7. This colchicine-injected tissue shows various degrees of sloughing in three cross-sectioned tubules. The Stage VII tubule on the right shows some sloughing of late (step 19) spermatids; the Stage VIII tubule at the upper left shows sloughing of step 8 spermatids; the Stage XIV tubule at the lower left shows sloughing of late (step 14) spermatids and meiotic arrest (compare with normal meiosis in Fig. 5). Step 6 and 18 spermatids (arrows) from another segment of the tubule are present within the lumen of the tubule. x 600. Fig. 8. This colchicine-injected tissue shows sloughing from two tubules. Above, both the early (step 7) and late (step 19) spermatids have been shed from an area of a Stage VII tubule. In another area of the tubule, the epithelium displays a full complement of germ cells. Late spermatids (step 19) have been sloughed into the lumen. Below, a Stage II tubule has lost all germinal cells except pachytene spermatocytes and mitotically arrested spermatogonia (arrow). Step 1 spermatids are seen within the lumen (arrowhead). x 700. Fig. 9. The seminiferous epithelium of two adjoining tubules shown in this vinblastine-treated tissue, has sloughed leaving only preleptolene spermatocytes (arrows) and the basal aspect of Sertoli cells containing their nuclei (arrowheads). x 1100. Figs. IO, 1 I. These vinblastine-treated tissues show sloughed cells in the tubular lumen. In Fig. 10, a cleft has formed between pachytene spermatocytes and step 7 spermatids (asterisk); and in Fig. 1 I, a separation of cellular elements (curved arrows) has taken place between like germ cell types (infrequently seen). Some Sertoli-germ cell associations protrude into the tubular lumen: but, in the same profile, remain attached at one end to the intact seminiferous epithelium. x 650, x 900. Figs. 12, 13. Electron micrographs, showing 6 hr (Fig. 12) and 12 hr (Fig. 13) post-injected
portions of seminiferous (colchicine) animals.
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Fig. 12. Only late spermatids, which are seen within the lumen, have been sloughed from this Stage VII tubule. Sertoli cell fragments surround the heads (isolated arrows) of these sloughed spermatids. The Sertoli cell may be identified by its characteristic (I) mitochondria, (2) endoplasmic reticulum, and (3) tubulobulbar complexes. The seminiferous epithelium shows delicate Sertoli processes which course between early (step 7) spermatids to reach the lumen (arrowheads). x 3500. Fig, atocytes well as cleaved arrows.
13. All cells shown except pachytene spermatocytes (ps), pre-leptolene sperm(~1s) and a few spermatids (s) have been sloughed. The lumen is indicated as possible ‘break points’ at which the Sertoli cell (S) may have been recently (straight arrows). The area of the blood-testis barrier is indicated by curved x 3200.
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plastic sections with the light microscope (Fig. 16). No sertoli nuclei were ever observed as part of a Sertoli cell fragment; consequently, it was assumed that only the apical portions of the Sertoli cells were cleaved from the seminiferous epithelium. Sertoli nuclei were always seen at the basal aspect of the Sertoli cells which remained along the tubular periphery. Sloughing of germ cells and Sertoli cells usually took place along a circumferential line which paralleled the limiting membrane and demarcated one germ cell generation from another (Figs. 3, 7, 8, 11). Thus, only late spermatids (Figs. 7, 11) might be sloughed; or both late and early spermatids (Figs. 7, 8); or more rarely, pachytene spermatocytes, early and late spermatids (similar to that shown in Fig. 9). A line of vacuoles or clefts which were present between dissimilar spermatogenic cells was often observed (Figs. 10, 11). These images all suggested intermediate steps in the
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process by which germ cells and Sertoli cells were sloughed. Fewer microtubules were present in Sertoli cells of treated testes compare Fig. 14 with Fig. 15). This was determined by subjective visual estimation where the plane of sectioning was perpendicular to the ‘trunk’ region of the Sertoli cell at a level between round spermatids. Microtubules running radially within the cell appeared in crosssectional profile. Treated testes usually displayed no microtubules, nor were there crystalline lattices like that commonly seen after vinblastine treatment (Bensch and Malawista, 1969). Twelve hours post-injection. The sloughing of tubular cells was more pronounced in the 12 hr post-injection samples. The lumina of many tubules were filled with fragments of sloughed Sertoli-germ cell associations (Fig. 3). The epithelium of some tubules was represented only by basal compartment cells (Russell, 1977a) and the very basal aspect
Figs. 14, 15. Stage VII tubules, in which the ‘trunk’ region of the Sertoli cell has been cross-sectioned as it lies between step 7 spermatids. All tissues are from near the site of injection (6 hr post-injection) and micrographs taken from the seminiferous tubules which showed a full complement of germinal cells. Fig. 14. This saline-injected tissue shows numerous microtubules cut perpendicularly to their direction of travel in the long axis of the Sertoli cell (S). x 20,000. Fig. 15. Colchicine-treated tissue showing an area comparable to that depicted in Fig. 14. Numerous mitochondria are present within the Sertoli cytoplasm (S) but no microtubules are found. x 30,000. Figs. 16-18. Micrographs of cell fragments present within the lumina of seminiferous tubules of colchicine-treated rats, sacrificed 6 hr after injection. Micrographs are taken, from tissues near the site of injection. Fig. 16. Light micrograph showing sloughed step 9 spermatids (arrows) which appear clustered around Sertoli fragments (arrowheads), some of which are intensely basophilic and others of which appear lightly stained. x 1200. Fig. 17. This Sertoli fragment (S) can be identified by its general relationship to the elongated step 19 spermatids (step 19), by the presence of Sertoli ectoplasmic specializations (es) and a tubulobulbar complex (tbc). Other nearby germ cells and Sertoli fragments are shown. x 9000. Fig. 18. Electron micrograph showing two Sertoli fragments (S) which appear in a rounded form. Step 8 spermatids are arranged in rosette fashion around these fragments attached over the acrosomal region to the Sertoli ectoplasmic specialization (arrows). Residual bodies (rb) have been internalized by the Sertoli cell prior to severance of the fragment from the body of the cell. x 4000.
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of the Sertoli cell which frequently extended no more than 8 pm in a centripetal direction (Fig. 9). Sertoli-Sertoli junctions were always present (Fig. 13). Long-term experiments. Saline-injected control testes, whether examined I2 hr postinjection or 60 days post-injection, were indistinguishable histologically and cytologically from normal, adult untreated rats. The response of 12 hr post-injection testes from colchicine and vinblastine injected animals used in the long-term experiments was identical, and was similar to the response of the I2 hr post-injected colchicine treated testes described in the previous paragraph. Similarly, the effects of colchicine and vinblastine in 60 day post-injection animals were also indistinguishable and will be described together. Examination of the 12 hr treatment tissues verified that extensive sloughing of apical Sertoli processes, with their attached germ cells, had occurred. The long-term (60 day) post-injection testis displayed severely depleted tubules (Fig. 19) with only occasional spermatogonia near the limiting membrane. Numerous Sertoli cells lined the small seminiferous tubules, often completely filling the lumen. Heterochromatin clumps were prominent, amassed along the inner aspect of the highly infolded nuclear envelope (Fig. 21). Sertoli cells demonstrated numerous finger-like processes which intervened between other nearby Sertoli cells (Figs. 20, 21). These processes displayed numerous microtubules and their surfaces showed junctional relationships to other nearby Sertoli cells (Fig. 20). Discussion Studies using microtubular disrupting agents in the testis have largely focused on the effect of these agents on spermiogenesis, with special attention paid to the effects of these drugs on the initial elongation phase of spermatids (Rattner, 1970; Wolosewick and Bryan, 1977; Handel, 1979; Fawcett et al., 1971). In the present study, no changes in sperm head morphology were detected. The fact that this did not occur possibly could be explained by inadequate penetration of these drugs through the blood-testis barrier (as discussed by Wolosewick and Bryan, 1977).
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Microtubules, in elongating mouse spermatids are disrupted if the germinal cells are placed in a cell culture medium and exposed to microtubular disrupting agents (Wolosewick and Bryan, 1977), thus allowing direct exposure of these agents to the germinal cells. Rattner (1970) and Handel (1979) reported defects in spermatid head morphology in the mouse and guinea pig respectively following intratesticular injections of colchicine. Our observations to the contrary do not mean that microtubular disrupting agents would not have affected these elongating cells, if given enough time to act; but this affect was possibly precluded by the rapid sloughing of such cells into the tubular along with Sertoli fragments. Manchette microtubules have been reported to be more resistant to microtubular disrupting agents (Wolosewick and Bryan, 1977). Not only was it difficult to test the role of microtubules in the elongation process in which spermatids attain their slender configurations, but for the same reasons it was not possible to test other postulated functions of microtubules. For example, Russell (1977b) has shown that microtubules are attached to cisternae of endoplasmic reticulum comprising the ectoplasmic specializations, a structure which overlies and is known to be bound to the heads of elongating spermatids (Sapsford, 1963; Russell, 1977b; Romrell and Ross, 1979; Russell et a/., 1980). Microtubules, by virtue of their well-established role in the development, and maintenance of the cytoarchitecture of cells (Porter, 1966; Bloom and Fawcett, 1975; Ham, 1974) might then influence the movements of the late spermatids (as proposed by Christensen, 1965; Fawcett, 1975; Russell, 1977b) by acting on the mantle (ectoplasmic specialization) which covers and binds the spermatid head. These changes in position of the spermatid are well documented, especially at the time that elongated spermatids are translocated from the deep recesses within the Sertoli cell to shallow recesses at the surface of the seminiferous epithelium in preparation for spermiation (Leblond and Clermont, 1952; Russell, 1977b; and others); but as mentioned above, the involvement of microtubules in this process could not be studied due to the massive and rapid sloughing of cells.
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Aoki (1980) has reported fragmentation of Sertoli processes and premature sperm release from Stages VII and VIII after microinjection of microtubular inhibitors into seminiferous tubules. We question whether this effect was to cause sperm release or was a consequence of the fragmentation of the apical Sertoli processes as reported in the present study. Aoki also reports that the treatments led to a complete disorganization of the seminiferous tubulea consequence also demonstrated in the present report. The primary objective of the present study was the elucidation of early detectable changes throughout the seminiferous epithelium, especially the relationships of Sertoli cells to surrounding germ cells and the influence of agents on Sertoli architecture. Prior to doing the experiments reported in this paper, we utilized systemic injections of colchicine in an attempt to affect testis structure. The dosages utilized were always ineffective in that no changes in testicular structure were observed. When higher doses were utilized, the animals invariably died as the result of the well-known toxic effects of colchicine. Therefore, injections of colchicine and vinblastine directly into the testes were attempted, although this was not a novel approach (Rattner, 1970; Handel, 1979; Aoki, 1980). By injecting with a very small needle and by avoiding blood vessels in the process, there were no adverse mechanical and/or disruptive effects. The morphological patterns seen after
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injection of either vinblastine or colchicine were identical. First, the Sertoli microtubules were notably absent from the Sertoli cytoplasm of treated animals after only 6 hr post-injection. The labile nature of Sertoli microtubules was reported in the work of Rattner (1970), Wolosewick and Bryan (1977) and Aoki (1980). Secondly, mitotic and meiotic divisions were arrested, an effect which would be anticipated due to the disruption of the spindle apparatus which usually occurs in treatments of this kind. Arrest of meiosis in the male using microtubular inhibitors, to our knowledge, has not been demonstrated previously. This indicates that the drugs under consideration are capable of penetrating the blood-testis barrier to reach adiuminal compartment cells (Russell, 1979b). Thirdly, portions of tubules showing Sertoli and closely related germ cells were sloughed leaving a variably denuded seminiferous epithelium. Vinblastine and colchicine are unrelated chemically, but they both have well-documented disruptive effects on microtubules. Although their mechanisms of action are not identical, these drugs bind selectively to tubulin subunits and disrupt the assembly of microtubules (Dustin, 1978). It is suggested that the effects seen here are due solely to the disruption of microtubules, as these cytoplasmic structures were clearly less numerous within the Sertoli cells of treated rats. Such a morphological pattern of response in the testis can be predicted regardless of the nature of the agent that might be used in the
Figs. 19-21. Tissues taken from rats 60 days after a single colchicine tissues were taken from near the site of injection.
injection.
All
Fig. 19. This light micrograph shows a tububular cross-section which displays only Sertoli cells and their numerous processes. Sertoli cells appear to completely fill the tubule and their nuclei are highly infolded. x 750. Fig. 20. High magnification micrograph showing a Sertoli process and the numerous microtubules which run in its long axis. x 40,000. Fig. 21. In this electron micrograph, Sertoli nuclei appear highly infolded and show numerous heterochromatin clumps postitioned along the inner aspect of the nuclear membrane. Many small cell processes intervene between the bodies of these Sertoli cells forming junctional contacts between them and neighboring Sertoli cells (arrows). x 10,000.
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future to disrupt microtubules. Another morphological pattern of response has also been shown. This takes place when the hormonal pathway leading to the stimulation of the testis is interrupted at one or more levels to prevent hormones from interacting with their target cells (production, release, binding, etc.; Russell et al., 1981). Clearly, the number of agents used to disrupt spermatogenesis is many times greater than the number of ‘vital pathways’ interrupted (Flickinger, 1976; Russell et al., 1981). The question of why Sertoli cells and germ cells are sloughed from the seminiferous tubule should be addressed. From semiserial section studies (Russell and Wong, 1981; Wong and Russell, 1981), the Sertoli cell was shown to be a highly asymmetrical cell, about two to three times longer (in a basalapical or centripetal dimension) than it was wide. Microtubules are known to be numerous and run in its long axis (Christensen, 1965; Fawcett, 1975). The Sertoli cell shows numerous attachment devices to the various types of germ cells which are associated with it (Russell, 1980). Microtubule disruption would result in the loss of ‘skeletal’ support of the Sertoli cell and would tend to destabilize the cell. When this happens, the cell would naturally tend to round-up or assume a less asymmetrical form, as has been shown for other cell systems exposed to microtubule destabilizing agents (Porter, 1966; Bessis and BretonGorius, 1967; Behnke, 1970). In attempting to do so, the Sertoli cell, as a whole, would not be able to assume a less asymmetrical shape in view of its attachments to numerous surrounding germinal cells. It seems logical that in attempting to round-up, the basal part of the cell would exert tension or pull on the apical Sertoli processess, and these in turn would break off from the body of the Sertoli cell at one or more points. Once broken, the apical portion of the cell would itself roundup and remain associated with the attached germinal elements. This hypothesis fits our observations, that many Sertoli fragments remained attached to nearby germ cells. This pattern of Sertoli-germ cell sloughing with concomitant rounding-up of the Sertoli cell is especially evident from figures such as 16 and 18. Here a rosette configuration is assumed with germ cells positioned peripherally about a large rounded Sertoli
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fragment, with the germ cell attachments occurring at the plasma membrane covering the Sertoli ectoplasmic specialization. Sertoli cells were usually sloughed in circumferential layers of like germ cells (the cleft between sloughed cells was between dissimilar cell types). This preferential sloughing appears likely in view of the integrity provided to like germ cell types by connecting intercellular bridges. Thus, clones of similar cell types were probably held together by these bridges during the sloughing process. Basal compartment cells were never shed into the tubular lumen. It should be mentioned that not all cell types responding to vinblastine or colchicine treatment respond to these drugs by changes in their form. There is clear evidence for a role of microtubules in development of cell shape (Tilney, 1971) but this role appears to be variable. For example, axopodial shape in Heliozoans is maintained by microtubules and is subject to the influence of microtubular destabilizing drugs (Tilney, 1968). However, in chick lens development, the sensitivity to colchicine is lost following elongation of the lens fiber cell (Downie and Pegrum, 1971). Thus, other factors such as filaments, junctions, etc., must act to stabilize some cells. Sertoli cells, unlike lens fiber cells, must continually change their configuration to accommodate the progressive upward movement of germ cells and the varied morphological shapes the germ cells take at different stages in the spermatogenic cycle (Russell, 1980; Russell and Wong, 1981). Thus the Sertoli cell, as shown in the present study, would be highly suspect as a cell type easily influenced by microtubular destabilizing agents. Microtubules ran radially within the Sertoli cell and did not enter the numerous lateral processes which intervene between round germ cell types (Russell and Wong, 1981) and other Sertoli cells (Russell, 1979b). It would be unreasonable to suggest that the formation and maintenance of elaborate lateral (and, possibly, some apical) processes, were all directly related to the activities of cytoplasmic microtubules. Microfilaments have been shown to be abundant within the Sertoli cell (Flickinger and Fawcett, 1976; Fawcett, 1975; Russell and Clermont, 1976) and these structures are undoubtedly important in a variety of shape changes
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occuring during the spermatogenic cycle (Fawcett, 1975; Russell, 1977a, b; Burton et al., 1980; Suarez-Quian ef al., 1980). It should be emphasized that it would be naive to suggest that only cell structures involved by the treatments were microtubules. Recent research clearly shows that other elements, especially the system of microfilament, interact with microtubules (Griffith, 1980; Pollard, 1980; Wolosewick and Porter, 1976). Although the primary effects noted in this study are probably due to microtubular destabilization, the relative roles of microtubules and microfilaments in development and maintenance of the configuration of the Sertoli cell and its processes await further elucidation. The intent of the long-term studies was to assess the regenerative capabilities of the Sertoli cells and spermatogenic cells after shedding and elimination of the majority of the apical Sertoli cell and its attached germinal cells. First, one injected testis was processed at 12 hr post-injection to predict the severity of the response in the testicle of the opposite side. Once it was determined that a massive sloughing response was present on one side, the other testicle was allowed to remain in situ for about one cycle of the
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seminiferous epithelium (Clermont rt a/., 1959). At sacrifice, this testis was small and largely depleted of germ cells. The tubule showed numerous Sertoli cells which were relatively small but, nevertheless, much larger than many of those Sertoli cells from tubules of the 12 hr testis which had undergone sloughing of their apical components to the level of the blood-testis barrier. The extent of the ability of these cells to regenerate their apical cytoplasmic processes remains in doubt since morphometric studies were not performed; but our subjective observations suggest that the Sertoli cells appear to have considerable ability to regenerate apical cytoplasm and repopulate their processes with microtubules. The necessity for the presence of a population of germ cells for the Sertoli cell to assume its typical configuration is underscored. Acknowledgements The use of the Springfield S.M. Electron Microscope Facility is acknowledged. Mr R. Venezia provided excellent technical assistance in this project supported by a special in-house (SIU) Research, Development and Administration Grant.
References AOKI, A. 1980. Induction of sperm release by microtubule inhibitors in rat testis. Eur. J. Cell Eiol.. 22, 467. BEHNKE, 0. 1970. Microtubules in disk-shaped blood cells. Znf. Rev. cxp. Path., 9, l-92. BENSCH, K. G. and MALAWISTA. S. E. 1969. Microtubular crystals in mammalian cells. J. Cell Biol., 40, 95-106.
BESSIS, M. and BRETON-GORIUS, J. 1967. Rapports entre noyau et centrioles dans les granulocytes iztalks. Rble des microtubules. Nouv. Rev. Fr. HPmatol., 7, 601-620. BLOOM, W. and FAWCETT, D. W. 1975. A Textbook of Histolog),, pp. 59-61. Saunders, Philadelphia. BURTON, P. R., DIENER, D. R. and FROST, L. C. 1980. Cytoskeleton of immature rat Sertoli cells in vitro. f. Cell Biol., 87, 213A. BYERS,B. and PORTER, K. R. 1964. Oriented microtubules in elongating cells of the developing lens rudiment after induction. Proc. not. Acad. Sci., U.S.A., 52, 1091-1099. CHRISTENSEN,A. K. 1965. Microtubules in Sertoli cells of guinea-pig testis. Anar. Ret,., 151, 335. CLERMONT.Y., LEBLOND, C. P. and MESSIER,B. 1959. Duree du cycle de I’epithelium stminal du rat. Awh. Amt.
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DOWNIE, J. R. and PEGRUM,S. M. 1971. Organization 26, 623-635. DUSTIN, P. 1978. Microtubules,
ofthe chick blastoderm
edge. J. Embryo/.
exp. Morph..
pp. 226-251. Springer-Verlag, New York. ELFTMAN, H. 1963. Sertoli cell and testis structure. Am. J. Annr., 113, 25-33. FAWCETT, D. W. 1975. Ultrastructure and function of the Sertoli cell. In Handbook ofPh.vsio/og)~ (ed. R. 0. Creep), Vol. V, pp. 21-55. Physiological Sot., Washington, D.C. FAWCETT, D. W. and PHILLIPS, D. M. 1969. Observations on the release of spermatozoa and on changes in the head during passage through the epididymis. J. Reprod. Fert., Suppl., 6, 405-418.
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FAWCETT, D. W., ANDERSON, W. A. and PHILLIPS, D. M. 197 I. Morphogenetic factors influencing the shape of the sperm head. Devl. Biol., 26, 220-251. FAWCETT, D. W. and WITEBSKY, F. 1964. Observations on the ultrastructure of nucleated erythrocytes and thrombocytes with particular reference to the structural basis of their discoidal shape. Z. Zellforsch. mikrosk. Anat., 62, 785-806. FLICKINGER, C. J. 1976. The influence of antifertility agents on the fine structure of the male reproductive tract. Proc. 34th Ann. EMSA Meeting, Miami, pp. 18-19. FLICKINGER, C. J. and FAWCETT, D. W. 1967. The junctional specializations of Sertoli cells in the seminiferous epithelium. Anal. Rec., 158, 207-222. GRAVIS, C. J. 1978. A scanning electron microscopic study of the Sertoli cell and spermiation in the Syrian hamster. Am. J. Anat., 151, 21-38. GRIFFITH, L. M. 1980. Viscometric analysis of the interaction of actin filaments with microtubules and with unfractionated or purified microtubule associated proteins. Eur. J. Cell Biol., 22, 290. HAM, A. W. 1974. Histology, pp. 141-144. J. B. Lippincott, Philadelphia. HANDEL, M. A. 1979. Effects of colchicine on spermiogenesis in the mouse. J. Embryo/. exp. Morph., 51, 73-83. 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. SC;., 55, 548-573. POLLARD, T. D. 1980. Association of contractile proteins with microtubules and their possible participation in microtubule dependent movements. Eur. J. CeN Biol., 22, 289. PORTER, K. P. 1966. Cytoplasmic microtubules and their functions: In Ciba Foundation Symposium in Principles of Bimolecular Organization, pp. 308-345. Churchill, London. RATTNER, J. B. 1970. Effects of colcimid on the ultrastructure of mammalian spermiogenesis. J. Cell Biol., 47, 168a. ROMRELL, L. J. and Ross, M. H. 1979. Characterization of Sertoli cell-germ cell junctional specializations in dissociated testicular cells. Anat. Rec., 193, 23-42. RUSSELL, L. D. 1977a. Movement of spermatocytes from the basal to the adluminal compartment of the rat testis. Am. J. Anat., 148, 313-328. RUSSELL, L. D. 1977b. Observations on rat Sertoli ectoplasmic (‘junctional’) specializations in their association with germ cells in the rat testis. Tissue & Cell, 9, 475-498. RUSSELL, L. D. 1979a. Further observations on tubulobulbar complexes formed by late spermatids and Sertoli cells in the rat testis. Anat. Rec., 194, 213-232. RUSSELL, L. D. 1979b. Observations on the inter-relationships of Sertoli cells at the level of the bloodtestis barrier: evidence for formation and resorption of Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle of the rat. Am. J. Anat., 155, 259-280. RUSSELL, L. D. 1980. Sertoli-germ cell interrelations: a review. Gamete Rex., 3, 179-202. RUSSELL, L. D. and BURGUET, S. 1977. Ultrastructure of Leydig cells as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture. Tissue & Cell, 9, 751-766. RUSSELL, L. D. and CLERMONT, Y. 1976. Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules. Anat. Rec., 185, 259-278. RUSSELL, L. D. and FRANK, B. 1978. Characterization of rat spermatocytes after plastic embedding. Arch. Androl., 1, 5-18. RUSSELL, L. D. and MALONE, J. P. 1980. A study of Sertoli-spermatid tubulobulbar complexes in selected mammals. Tissue & Cell, 12, 263-285. RUSSELL,L. D. MALONE, J. P. and KARPAS, S. 1981. Morphological patterns elicited following administration of agents known to affect spermatogenesis by disruption of its hormonal stimulation. Tissue & Cell, 13, 369-379. RUSSELL, L. D., MYERS, P., OSTENBURF, J. and MALONE, J. 1980. Sertoli ectoplasmic specializations during spermatogenesis. In Testicular Development, Structure and Function (eds. A. Steinberger and E. Steinberger), pp. 55-63. Raven Press, New York. RUSSELL, L. D. and WONG, V. 1981. Three-dimensional reconstruction of a Stage V rat Sertoli cell: size, configuration, and general relationship to germ cell. J. Androl., 2, 24-25. SAPSFORD, C. S., 1963. The development of the Sertoli cell of the rat and mouse; its existence as a mononucleate unit. J. Anat., 97, 225-238. SUAREZ-QUIAN, C. A., LIN, Y. C., BEGG, D. A., FUJIWARA, K. and DYM, M. 1980. The role of cytoplasmic filaments in cultured Sertoli cells. J. Cell Biol., 87, 151a. TILNEY, L. G. 1968. Studies on microtubules in Heliozoa. IV. The effect of colchicine on the formation and maintenance of axopodia and the redevelopment of pattern in Actinosphaerium nucIeofi/um (Barrett). J. Cell SC;., 3, 549-562. TILNEY, L. G. 1971. Origin and continuity of microtubules. In Origin and Continuity of Cell Organel/es (eds. Reinert and Ursprung), Vol. 2, pp. 222-260. Springer-Verlag, New York.
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TILNEY, L. G. and PORTER, K. R., 1965. Studies on the microtubules of Heliozoa. 1. Fine structure in Actinosphaerium with particular reference to enamel rod structure. Protop/asma, 50, 317-344. VITALE, R., FAWCETT, D. W. and DYM, M. 1973. The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment. Anar. Rec., 176, 333-344. WOLOSEWICK, J. J. and BRYAN, J. H. D. 1977. Ultrastructural characterization of the manchette microtubules in the seminiferous epithelium of the mouse. Am. J. Amt., 150, 301-332. WOLOSEWICK, J. J. and PORTER, R. R. 1976. Stereo high-voltage electron microscopy of whole cells of the human diploid line. Am. J. Anar. 147, 303-324. WONG, V. and RUSSELL, L. D. 1981. Three-dimensional reconstruction of a Stage V rat Sertoli cell: methodological considerations. Amt. Rec.. 199, 281a.
LONNIE D. RUSSELL, JAMES P. MALONE and DANIEL S. MacCURDY
EFFECT OF THE MICROTUBULE DISRUPTING AGENTS, COLCHICINE AND VINBLASTINE, ON SEMINIFEROUS TUBULE STRUCTURE IN THE RAT ABSTRACT, Injections of colchicine or vinblastine were given intratesticularly and rats sacrificed 6 and 12 hr later. Colchjcine and vinblastine produced identical morphological patterns of response in the seminiferous tubules resulting in arrest of germcell mitoses and meioses and a rapid depletion of the microtubules normally found within the Sertoli cell. Sloughing of cells into the lumen of seminiferous tubules was the most prominent feature noted. Germ cells and portions of the apical Sertoli cells were frequently sloughed together where they remained in close association. Usually germ cells and associated Sertoli cell fragments were cleaved from the wall of the seminiferous tubule at a level between dissimilar generations of germ cells, e.g. between spermatocytes and spermatids. This selective sloughing probably occurred as the result of the support normally provided by intercellular bridges which link clones of like germ cell types. Sequential steps in the process leading to sioughing of Sertoli-germ cell associations could be inferred from observations made in plastic 1 pm sections. Cell sloughing at 12 hr post-injection was generally more extensive. It was frequently noted that germ cells and the apical portions of Sertoli celts had been extruded to the level of the most adluminal tight junctions forming the blood-testis barrier. It was concluded that disruption of Sertoli microtubules was responsible for sloughing of Sertoli fragments and associated germ cells, and that the cytoskeletal support of the Sertoli cell was, at least in part, dependent upon the integrity of Sertoli microtubules. The Sertoli cell could not round-up after loss of its cytoskeletal support, due to the numerous attachment devices known to link it with various apically positioned germ cells. Thus, the cell was severed at some point along its delicate apical processes, as the consequence of forces produced by the ‘rounding-up’ process. Long-term sacrifice after vinblastine or colchicine treatment allowed the Sertoli cells to regain microtubules and long processes but not their typical configuration. Spermatogenesis remained severely impaired.
Introduction
Until recently, its three-dimensional configuration had not been realized (Wong and Russell, 1981; Russell and Wong, 1981). These studies employed reconstruction techniques to make a Sertoli cell model. The Stage V reconstructed Sertoli cell extended from the base of the seminiferous tubule to the tubular lumen and, although irregularly shaped, could generally be regarded as a columnar cell. The reconstructed model represented only one of two major architectural forms of the Sertoli cell, since the Sertoli cell is thought to change its basic configuration in
OF all the cells in the body, the Sertoli cell is one of the most complex, both from a structural and a functional standpoint (Fawcett, 1975; Russell, 1980). The fine details of its in vivo shape have long been hypothesized, based on evidence provided by studies employing random thin sections. Department of Physiology and School of Medicine, Southern Illinois University, Carbondale, IL 62901. Received 6 November 1980. Revised 7 February 198 I. 349
350 stages (VII-VIII) of the spermatogenic cycle which prepare the sperm for its eventual release (Fawcett and Phillips, 1969; Russell and Clermont, 1976; Gravis 1978). Throughout the cycle, the Sertoli cell closely conforms to the outlines of the germinal cells and, in certain regions, to other Sertoli cells. By virtue of its configurational relationships to nearby germ cells, it has long been hypothesized to be important in the development and release of these cells (Fawcett, 1975; Russell, 1980). Microtubules have generally been considered as important structures in the maintenance of cell architecture (asymmetrical cell shape) such as that displayed by the Sertoli cell. They do so by acting as semi-rigid skeletal elements supporting (Byers and Porter, 1964; Fawcett and Witebski, 1964; Tilney and Porter, 1965; and many others) although they may act in concert with the system of microfilaments or other support elements (Pollard, 1980; Griffith, 1980; Wolosewick and Porter, 1976). A large body of evidence showing that microtubules are important in the attainment and/or maintenance of cell shape is derived from studies in which the destruction of microtubules has led to a change in cell shape and/or studies which have demonstrated a preferential orientation of microtubules in the long axis of a cell or cell process (Dustin, 1978; Ham, 1974; Fawcett, 1975). Christensen (1965) first described microtubules within the Sertoli cell of the guineapig as being abundant and extending radially within the cell (from the base to the lumen). This particular orientation of microtubules in the long axis of the Sertoli cell has been confirmed by Fawcett (1975). Christensen felt that microtubules ‘may support the tenuous cytoplasmic extensions of the Sertoli cells, and produce the various movements that spermatids undergo in the epithelium over the course of spermatogenesis. These concepts have never been tested experimentally, although we note the work of Wolosewick and Bryan (1977) and Handel (1979) who studied the effects of microtubule disrupting agents on spermiogenesis in the mouse, and Rattner (1970) who studied the effects of colchicine on In these spermiogenesis in guinea-pigs. studies, effects on Sertoli cells were not
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specifically examined; however, after treating mice with colchicine, Handel (1979) noted vacuolization of the Sertoli cell and apical Sertoli processes bulging into the tubular lumen. Wolosewick and Bryan (1977) and Rattner (1970) remarked on the rapid disappearances of Sertoli cell microtubules subsequent to treatment with microtubule destabilizing drugs. A very recent report (Aoki, 1980) describes the effects of microtubular agents injected into the lumina of the seminiferous tubules. Early changes including a fragmentation and retraction of Sertoli cell processes were suggested to be responsible for premature sperm release. In the present study, the microtubular disrupting agents, vinblastine and colchicine, were used for the primary purpose of observing the short-term changes in the Sertoli cell with special attention paid to changes related to its configuration. When administered appropriately, these drugs caused marked alterations in Sertoli-cell architecture which led to massive disruption of spermatogenesis. Materials and Methods Injections Twelve adult, male, Sprague-Dawley rats weighing 250-300 g were immobilized and one testis was injected, using a 32 gauge needle, with either colchicine (5 x 10m5 mg in 0.5 ml normal saline) or vinblastine (2.5 x 10m5 mg in 0.5 ml normal saline). Six control rats were given saline alone (0.5 ml). Prior to injection, the scrotal skin was shaved and cleaned with a generous amount of 70% ethanol. Under strong light, the tortuous aspect of the testicular artery was revealed. The juncture of the caudal third with the middle third of the testis was injected without rupturing blood vessels. Injections were made into the central area of the testis. Tissue preparation One-half of the animals in treatment and control groups were sacrificed six hr postinjection with the other half at 12 hr; and the tissue prepared for light and electron microscopic observation. To do this, animals were anesthesized with Nembutal and the testis perfused-fixed for 45 min with 5% gluteraldehyde in 0.2 M caccodylate buffer (ph 7.4) by a retrograde abdominal aorta
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method (Vitale et al., 1973). Subsequently, testis tissue within 3 mm of the injection site (as roughly estimated) and at the proximal pole of the testis were diced into small cubes and immersed in the same fixative solution for 30 min. Post-fixation was performed for 1 hr employing an osmium: ferrocyanide mixture (Russell and Burguet, 1977). After a brief wash in buffer, tissues were subsequently dehydrated, infiltrated and embedded in Araldite. Blocks were oriented such that round or nearly round (cross-sections) and longitudinal sections of tubules would be produced upon sectioning at 1 pm with an MT-1 Porter-Blum ultramicrotome. Thin sections of silver and silver-gold interference colors were obtained with a Sorvall MT-2 ultramicrotome and examined on a Philips 201 electron microscope. Long-term
experiments
For long-term experiments, three rats were injected with either vinblastine or colchicine at the specific dosages and volume of vehicle described above. Three control rats were injected with saline only. At twelve hours post-injection, the right testis was surgically removed and processed for light and electron microscopic observation. Perfusion fixation was accomplished with a 26-gauge needle inserted into the testicular artery. All rats were sacrificed sixty days later, whereupon the left testis was removed and perfusedfixed through the testicular artery. Tissues were prepared in a manner similar to that described above for the short-term experiments. Results Safine controk Animals injected with saline showed normal testicular structure at each of the 6 and 12 hr sacrifice intervals selected (Fig. 1). There was no evidence of hemorrhage in the intertubular spaces or of disruption or injury to the seminiferous tubules. Germ cells remained in their typical position (Russell, 1980) within the seminiferous epithelium. Microtubules were distributed throughout the Sertoli cell, but were scarce in the region lateral to and below the Sertoli nucleus (near the limiting membrane). In the supranuclear region, microtubules were more numerous and were oriented radially (in the long axis of the Sertoli cell), as indicated by
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Christensen (1965) and Fawcett (1975). In all stages, except VII and VIII (classification of Leblond and Clermont, 1952), microtubules coursed alongside elongating and elongate spermatids which were positioned in deep recesses within the Sertoli cell. Microtubules were also seen in close relationship to cytoplasmic vesicles (Fawcett et al., 1971) and Sertoli ectoplasmic specializations (Russell, 1977b). In Stages VII and VIII, they extended within the ‘trunk’ of the Sertoli cell (see Elftman’s (1963) comparison of the form of a Sertoli cell to that of a tree) and into the small processes which SUJrounded the heads of late spermatids (Russell, 1979a; Russell and Malone, 1980). When the trunk region of the Sertoli ceil was CJOSSsectioned (sections obtained from longitudinally oriented tubules within tissue blocks), this thickened portion of the cell appeared as shown in Fig. 14. It displayed numerous microtubules, also cut in CJOSSsection, which traveled in the long axis of the cell. Mitotic and meiotic divisions occurred in a typical and predictable pattern as has been described from numerous kinetic studies. A few mitoses of spermatogonia were seen at the periphery of a seminiferous tubule in which divisions were known to OCCUJ. In Stage XIV, primary spermatocytes are known to undergo two rapid successive cytokineses (Russell and Frank, 1978). This cell division pattern was also observed in saline injected animals (Fig. 5). Treated animals Animals injected with either colchicine or vinblastine appeared to show identical responses. Consequently, the following description applies to testes treated with either agent. Six hours post-injection. Tissues within 3 mm of the injection site showed an identical response, both in terms of the pattern of response and the magnitude of response, to those taken more distally. Mitotic (Fig. 4) and meiotic (Figs. 6, 7) arrest was a common feature. Arrest of spermatogonial divisions was frequently seen (Fig. 4) and the arrested cells usually ringed the entire tubule. Numerous spermatocytes arrested at meiosis I and II were present in Stage XIV and I tubules. Many of these appeared degenera-
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tive and showed a marked basophillia with distortion and clumping of nuclear cytoplasmic components (Fig. 6). Spermatids undergoing early phases of elongation in Stages VIII, IX and X were apparently unaffected by the treatments. Microtubules were present and appeared to form and/or maintain a more or less typical manchette. The most conspicuously abnormal feature within the seminiferous tubules was the sloughing of germinal and non-germinal elements. Some tubules showed no evidence of cell sloughing (Fig. 2); but in most, this process took place to a variable degree (Figs. 2, 7, 8, 10, 12). Not only were germ cells shed, but the apical portions of Sertoli cells attached or related to many of these germ cells were broken away from their parent cells. Evidence for this process came from both light and electron microscopy, where it was observed that whole segments of the seminiferous epithelium appeared to be sloughed. This appeared to occur by a mechanism which involved cleavage of the Sertoli cell at some point above its nucleus (apical aspect). These sloughed Sertoli cellgerm cell associations were then freed into the tubular lumen (Figs. 2, 3, 7, 8, 10, 12, 16-l 8). Sloughed Sertoli cell fragments within the tubular lumen were frequently observed in the form of large rounded bodies indicating that the previously columnar aspect of the apical Sertoli cell had rounded-up (Figs. 16-18) during and/or after the process
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of being sloughed. Elongated spermatids were frequently observed within recesses of these rounded Sertoli profiles (Fig. 17). Germ cells undergoing the elongation process were arranged in ‘rosettes’ around a centrally positioned round Sertoli cell fragment (Figs. 16, 18). Sertoli fragments often appeared degenerate, showing ruptured membranes and some loss of cytoplasmic matrix (Fig. 17). Areas where breaks might have occurred between the parent Sertoli cell and the Sertoli fragment were difficult to identify; however, observations represented by Fig. 13 (12 hr post-injection rat) suggested recent separation of apical Sertoli processes. The apical processes in affected tubules were typically seen as tenuous extensions between the round germ cells which remained to line the tubular lumen (Fig. 12). Occasionally, Sertoli processes reached the lumen; but, large ‘trunks’ of Sertoli cells which had presumably undergone apical separation, appeared to have retracted except in regions where the epithelium was severely depleted. Sertoli cell fragments could be identified with the electron microscope by using characteristic ultrastructural features such as the presence of tubulobulbar complexes (Russell and Clermont, 1976; Figs. 12, 17), ectoplasmic specializations (Flickinger and Fawcett, 1967; Russell, 1977b; Figs. 17, 18), and the appearance of the mitochondria (Fig. 12). Once identified at the electron microscope level, large Sertoli fragments easily identified in 1 pm
Figs. 1-3. Light micrographs showing several cross-sectional profiles of seminiferous tubules, all of which were processed from an area near the site of injection. x 200. Fig. 1.This 6 hr, saline-injected control demonstrates a full complement of germinal cells and a typical association of cells forming the wall of the seminiferous epithelium. No cellular elements have been sloughed into the tubular lumen, and no hemorrhage is indicated in the intertubular spaces. Fig. 2. This colchicine-injected, 6 hr sacrificed animal shows regions of focal sloughing (arrows) and the presence of cellular debris in the lumina of some seminiferous tubules. Step 8 spermatids (arrowheads) are present in a Stage XIII tubule. Fig. 3. This colchicine-injected, 12 hr sacrificed animal shows massive sloughing of cellular elements as evidenced by depletion of cells from the seminiferous epithelium and the presence of loose cells within the tubular lumina. One tubule (at bottom center) shows sloughing of cellular elements, up to the region near the limiting membrane, leaving only one layer of germinal cells present (arrows).
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Figs. 4-6. Light micrographs of mitotic and meiotic cells, all of which were taken from tissues near the site of injection which have been sacrificed 6 hr post-injection. Fig. 4. This Stage VI seminiferous tubule, from a vinblastine-treated rat, was sectioned in a region where the tubule makes a bend. It shows several arrested spermatogonia in the process of dividing to form preleptolene spermatocytes (arrows). x 1250. Fig. 5. This Stage XIV (on the right) tubule from a saline-injected shows the typical appearance of dividing meiosis 11 cells. x 1100.
control
testis
Fig. 6. This Stage XIV tubule from a colchicine-injected testis shows the appearance of dividing meiotic cells, some of which appear atypical (arrows), others of which demonstrate astrong basophilia and are degenerating (arrowheads). Sloughing of some late spermatids (step 14) has occurred. x 1300. Figs. 771 I. Light micrographs of seminiferous tubules near the site of injection which have undergone various degrees of sloughing of germinal and Sertoli elements. Except for Fig. 9, which was taken from tissue prepared 12 hr post-injection, all photographs are from 6 hr post-injection animals. Fig. 7. This colchicine-injected tissue shows various degrees of sloughing in three cross-sectioned tubules. The Stage VII tubule on the right shows some sloughing of late (step 19) spermatids; the Stage VIII tubule at the upper left shows sloughing of step 8 spermatids; the Stage XIV tubule at the lower left shows sloughing of late (step 14) spermatids and meiotic arrest (compare with normal meiosis in Fig. 5). Step 6 and 18 spermatids (arrows) from another segment of the tubule are present within the lumen of the tubule. x 600. Fig. 8. This colchicine-injected tissue shows sloughing from two tubules. Above, both the early (step 7) and late (step 19) spermatids have been shed from an area of a Stage VII tubule. In another area of the tubule, the epithelium displays a full complement of germ cells. Late spermatids (step 19) have been sloughed into the lumen. Below, a Stage II tubule has lost all germinal cells except pachytene spermatocytes and mitotically arrested spermatogonia (arrow). Step 1 spermatids are seen within the lumen (arrowhead). x 700. Fig. 9. The seminiferous epithelium of two adjoining tubules shown in this vinblastine-treated tissue, has sloughed leaving only preleptolene spermatocytes (arrows) and the basal aspect of Sertoli cells containing their nuclei (arrowheads). x 1100. Figs. IO, 1 I. These vinblastine-treated tissues show sloughed cells in the tubular lumen. In Fig. 10, a cleft has formed between pachytene spermatocytes and step 7 spermatids (asterisk); and in Fig. 1 I, a separation of cellular elements (curved arrows) has taken place between like germ cell types (infrequently seen). Some Sertoli-germ cell associations protrude into the tubular lumen: but, in the same profile, remain attached at one end to the intact seminiferous epithelium. x 650, x 900. Figs. 12, 13. Electron micrographs, showing 6 hr (Fig. 12) and 12 hr (Fig. 13) post-injected
portions of seminiferous (colchicine) animals.
tubules
from
Fig. 12. Only late spermatids, which are seen within the lumen, have been sloughed from this Stage VII tubule. Sertoli cell fragments surround the heads (isolated arrows) of these sloughed spermatids. The Sertoli cell may be identified by its characteristic (I) mitochondria, (2) endoplasmic reticulum, and (3) tubulobulbar complexes. The seminiferous epithelium shows delicate Sertoli processes which course between early (step 7) spermatids to reach the lumen (arrowheads). x 3500. Fig, atocytes well as cleaved arrows.
13. All cells shown except pachytene spermatocytes (ps), pre-leptolene sperm(~1s) and a few spermatids (s) have been sloughed. The lumen is indicated as possible ‘break points’ at which the Sertoli cell (S) may have been recently (straight arrows). The area of the blood-testis barrier is indicated by curved x 3200.
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plastic sections with the light microscope (Fig. 16). No sertoli nuclei were ever observed as part of a Sertoli cell fragment; consequently, it was assumed that only the apical portions of the Sertoli cells were cleaved from the seminiferous epithelium. Sertoli nuclei were always seen at the basal aspect of the Sertoli cells which remained along the tubular periphery. Sloughing of germ cells and Sertoli cells usually took place along a circumferential line which paralleled the limiting membrane and demarcated one germ cell generation from another (Figs. 3, 7, 8, 11). Thus, only late spermatids (Figs. 7, 11) might be sloughed; or both late and early spermatids (Figs. 7, 8); or more rarely, pachytene spermatocytes, early and late spermatids (similar to that shown in Fig. 9). A line of vacuoles or clefts which were present between dissimilar spermatogenic cells was often observed (Figs. 10, 11). These images all suggested intermediate steps in the
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process by which germ cells and Sertoli cells were sloughed. Fewer microtubules were present in Sertoli cells of treated testes compare Fig. 14 with Fig. 15). This was determined by subjective visual estimation where the plane of sectioning was perpendicular to the ‘trunk’ region of the Sertoli cell at a level between round spermatids. Microtubules running radially within the cell appeared in crosssectional profile. Treated testes usually displayed no microtubules, nor were there crystalline lattices like that commonly seen after vinblastine treatment (Bensch and Malawista, 1969). Twelve hours post-injection. The sloughing of tubular cells was more pronounced in the 12 hr post-injection samples. The lumina of many tubules were filled with fragments of sloughed Sertoli-germ cell associations (Fig. 3). The epithelium of some tubules was represented only by basal compartment cells (Russell, 1977a) and the very basal aspect
Figs. 14, 15. Stage VII tubules, in which the ‘trunk’ region of the Sertoli cell has been cross-sectioned as it lies between step 7 spermatids. All tissues are from near the site of injection (6 hr post-injection) and micrographs taken from the seminiferous tubules which showed a full complement of germinal cells. Fig. 14. This saline-injected tissue shows numerous microtubules cut perpendicularly to their direction of travel in the long axis of the Sertoli cell (S). x 20,000. Fig. 15. Colchicine-treated tissue showing an area comparable to that depicted in Fig. 14. Numerous mitochondria are present within the Sertoli cytoplasm (S) but no microtubules are found. x 30,000. Figs. 16-18. Micrographs of cell fragments present within the lumina of seminiferous tubules of colchicine-treated rats, sacrificed 6 hr after injection. Micrographs are taken, from tissues near the site of injection. Fig. 16. Light micrograph showing sloughed step 9 spermatids (arrows) which appear clustered around Sertoli fragments (arrowheads), some of which are intensely basophilic and others of which appear lightly stained. x 1200. Fig. 17. This Sertoli fragment (S) can be identified by its general relationship to the elongated step 19 spermatids (step 19), by the presence of Sertoli ectoplasmic specializations (es) and a tubulobulbar complex (tbc). Other nearby germ cells and Sertoli fragments are shown. x 9000. Fig. 18. Electron micrograph showing two Sertoli fragments (S) which appear in a rounded form. Step 8 spermatids are arranged in rosette fashion around these fragments attached over the acrosomal region to the Sertoli ectoplasmic specialization (arrows). Residual bodies (rb) have been internalized by the Sertoli cell prior to severance of the fragment from the body of the cell. x 4000.
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of the Sertoli cell which frequently extended no more than 8 pm in a centripetal direction (Fig. 9). Sertoli-Sertoli junctions were always present (Fig. 13). Long-term experiments. Saline-injected control testes, whether examined I2 hr postinjection or 60 days post-injection, were indistinguishable histologically and cytologically from normal, adult untreated rats. The response of 12 hr post-injection testes from colchicine and vinblastine injected animals used in the long-term experiments was identical, and was similar to the response of the I2 hr post-injected colchicine treated testes described in the previous paragraph. Similarly, the effects of colchicine and vinblastine in 60 day post-injection animals were also indistinguishable and will be described together. Examination of the 12 hr treatment tissues verified that extensive sloughing of apical Sertoli processes, with their attached germ cells, had occurred. The long-term (60 day) post-injection testis displayed severely depleted tubules (Fig. 19) with only occasional spermatogonia near the limiting membrane. Numerous Sertoli cells lined the small seminiferous tubules, often completely filling the lumen. Heterochromatin clumps were prominent, amassed along the inner aspect of the highly infolded nuclear envelope (Fig. 21). Sertoli cells demonstrated numerous finger-like processes which intervened between other nearby Sertoli cells (Figs. 20, 21). These processes displayed numerous microtubules and their surfaces showed junctional relationships to other nearby Sertoli cells (Fig. 20). Discussion Studies using microtubular disrupting agents in the testis have largely focused on the effect of these agents on spermiogenesis, with special attention paid to the effects of these drugs on the initial elongation phase of spermatids (Rattner, 1970; Wolosewick and Bryan, 1977; Handel, 1979; Fawcett et al., 1971). In the present study, no changes in sperm head morphology were detected. The fact that this did not occur possibly could be explained by inadequate penetration of these drugs through the blood-testis barrier (as discussed by Wolosewick and Bryan, 1977).
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Microtubules, in elongating mouse spermatids are disrupted if the germinal cells are placed in a cell culture medium and exposed to microtubular disrupting agents (Wolosewick and Bryan, 1977), thus allowing direct exposure of these agents to the germinal cells. Rattner (1970) and Handel (1979) reported defects in spermatid head morphology in the mouse and guinea pig respectively following intratesticular injections of colchicine. Our observations to the contrary do not mean that microtubular disrupting agents would not have affected these elongating cells, if given enough time to act; but this affect was possibly precluded by the rapid sloughing of such cells into the tubular along with Sertoli fragments. Manchette microtubules have been reported to be more resistant to microtubular disrupting agents (Wolosewick and Bryan, 1977). Not only was it difficult to test the role of microtubules in the elongation process in which spermatids attain their slender configurations, but for the same reasons it was not possible to test other postulated functions of microtubules. For example, Russell (1977b) has shown that microtubules are attached to cisternae of endoplasmic reticulum comprising the ectoplasmic specializations, a structure which overlies and is known to be bound to the heads of elongating spermatids (Sapsford, 1963; Russell, 1977b; Romrell and Ross, 1979; Russell et a/., 1980). Microtubules, by virtue of their well-established role in the development, and maintenance of the cytoarchitecture of cells (Porter, 1966; Bloom and Fawcett, 1975; Ham, 1974) might then influence the movements of the late spermatids (as proposed by Christensen, 1965; Fawcett, 1975; Russell, 1977b) by acting on the mantle (ectoplasmic specialization) which covers and binds the spermatid head. These changes in position of the spermatid are well documented, especially at the time that elongated spermatids are translocated from the deep recesses within the Sertoli cell to shallow recesses at the surface of the seminiferous epithelium in preparation for spermiation (Leblond and Clermont, 1952; Russell, 1977b; and others); but as mentioned above, the involvement of microtubules in this process could not be studied due to the massive and rapid sloughing of cells.
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Aoki (1980) has reported fragmentation of Sertoli processes and premature sperm release from Stages VII and VIII after microinjection of microtubular inhibitors into seminiferous tubules. We question whether this effect was to cause sperm release or was a consequence of the fragmentation of the apical Sertoli processes as reported in the present study. Aoki also reports that the treatments led to a complete disorganization of the seminiferous tubulea consequence also demonstrated in the present report. The primary objective of the present study was the elucidation of early detectable changes throughout the seminiferous epithelium, especially the relationships of Sertoli cells to surrounding germ cells and the influence of agents on Sertoli architecture. Prior to doing the experiments reported in this paper, we utilized systemic injections of colchicine in an attempt to affect testis structure. The dosages utilized were always ineffective in that no changes in testicular structure were observed. When higher doses were utilized, the animals invariably died as the result of the well-known toxic effects of colchicine. Therefore, injections of colchicine and vinblastine directly into the testes were attempted, although this was not a novel approach (Rattner, 1970; Handel, 1979; Aoki, 1980). By injecting with a very small needle and by avoiding blood vessels in the process, there were no adverse mechanical and/or disruptive effects. The morphological patterns seen after
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injection of either vinblastine or colchicine were identical. First, the Sertoli microtubules were notably absent from the Sertoli cytoplasm of treated animals after only 6 hr post-injection. The labile nature of Sertoli microtubules was reported in the work of Rattner (1970), Wolosewick and Bryan (1977) and Aoki (1980). Secondly, mitotic and meiotic divisions were arrested, an effect which would be anticipated due to the disruption of the spindle apparatus which usually occurs in treatments of this kind. Arrest of meiosis in the male using microtubular inhibitors, to our knowledge, has not been demonstrated previously. This indicates that the drugs under consideration are capable of penetrating the blood-testis barrier to reach adiuminal compartment cells (Russell, 1979b). Thirdly, portions of tubules showing Sertoli and closely related germ cells were sloughed leaving a variably denuded seminiferous epithelium. Vinblastine and colchicine are unrelated chemically, but they both have well-documented disruptive effects on microtubules. Although their mechanisms of action are not identical, these drugs bind selectively to tubulin subunits and disrupt the assembly of microtubules (Dustin, 1978). It is suggested that the effects seen here are due solely to the disruption of microtubules, as these cytoplasmic structures were clearly less numerous within the Sertoli cells of treated rats. Such a morphological pattern of response in the testis can be predicted regardless of the nature of the agent that might be used in the
Figs. 19-21. Tissues taken from rats 60 days after a single colchicine tissues were taken from near the site of injection.
injection.
All
Fig. 19. This light micrograph shows a tububular cross-section which displays only Sertoli cells and their numerous processes. Sertoli cells appear to completely fill the tubule and their nuclei are highly infolded. x 750. Fig. 20. High magnification micrograph showing a Sertoli process and the numerous microtubules which run in its long axis. x 40,000. Fig. 21. In this electron micrograph, Sertoli nuclei appear highly infolded and show numerous heterochromatin clumps postitioned along the inner aspect of the nuclear membrane. Many small cell processes intervene between the bodies of these Sertoli cells forming junctional contacts between them and neighboring Sertoli cells (arrows). x 10,000.
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future to disrupt microtubules. Another morphological pattern of response has also been shown. This takes place when the hormonal pathway leading to the stimulation of the testis is interrupted at one or more levels to prevent hormones from interacting with their target cells (production, release, binding, etc.; Russell et al., 1981). Clearly, the number of agents used to disrupt spermatogenesis is many times greater than the number of ‘vital pathways’ interrupted (Flickinger, 1976; Russell et al., 1981). The question of why Sertoli cells and germ cells are sloughed from the seminiferous tubule should be addressed. From semiserial section studies (Russell and Wong, 1981; Wong and Russell, 1981), the Sertoli cell was shown to be a highly asymmetrical cell, about two to three times longer (in a basalapical or centripetal dimension) than it was wide. Microtubules are known to be numerous and run in its long axis (Christensen, 1965; Fawcett, 1975). The Sertoli cell shows numerous attachment devices to the various types of germ cells which are associated with it (Russell, 1980). Microtubule disruption would result in the loss of ‘skeletal’ support of the Sertoli cell and would tend to destabilize the cell. When this happens, the cell would naturally tend to round-up or assume a less asymmetrical form, as has been shown for other cell systems exposed to microtubule destabilizing agents (Porter, 1966; Bessis and BretonGorius, 1967; Behnke, 1970). In attempting to do so, the Sertoli cell, as a whole, would not be able to assume a less asymmetrical shape in view of its attachments to numerous surrounding germinal cells. It seems logical that in attempting to round-up, the basal part of the cell would exert tension or pull on the apical Sertoli processess, and these in turn would break off from the body of the Sertoli cell at one or more points. Once broken, the apical portion of the cell would itself roundup and remain associated with the attached germinal elements. This hypothesis fits our observations, that many Sertoli fragments remained attached to nearby germ cells. This pattern of Sertoli-germ cell sloughing with concomitant rounding-up of the Sertoli cell is especially evident from figures such as 16 and 18. Here a rosette configuration is assumed with germ cells positioned peripherally about a large rounded Sertoli
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fragment, with the germ cell attachments occurring at the plasma membrane covering the Sertoli ectoplasmic specialization. Sertoli cells were usually sloughed in circumferential layers of like germ cells (the cleft between sloughed cells was between dissimilar cell types). This preferential sloughing appears likely in view of the integrity provided to like germ cell types by connecting intercellular bridges. Thus, clones of similar cell types were probably held together by these bridges during the sloughing process. Basal compartment cells were never shed into the tubular lumen. It should be mentioned that not all cell types responding to vinblastine or colchicine treatment respond to these drugs by changes in their form. There is clear evidence for a role of microtubules in development of cell shape (Tilney, 1971) but this role appears to be variable. For example, axopodial shape in Heliozoans is maintained by microtubules and is subject to the influence of microtubular destabilizing drugs (Tilney, 1968). However, in chick lens development, the sensitivity to colchicine is lost following elongation of the lens fiber cell (Downie and Pegrum, 1971). Thus, other factors such as filaments, junctions, etc., must act to stabilize some cells. Sertoli cells, unlike lens fiber cells, must continually change their configuration to accommodate the progressive upward movement of germ cells and the varied morphological shapes the germ cells take at different stages in the spermatogenic cycle (Russell, 1980; Russell and Wong, 1981). Thus the Sertoli cell, as shown in the present study, would be highly suspect as a cell type easily influenced by microtubular destabilizing agents. Microtubules ran radially within the Sertoli cell and did not enter the numerous lateral processes which intervene between round germ cell types (Russell and Wong, 1981) and other Sertoli cells (Russell, 1979b). It would be unreasonable to suggest that the formation and maintenance of elaborate lateral (and, possibly, some apical) processes, were all directly related to the activities of cytoplasmic microtubules. Microfilaments have been shown to be abundant within the Sertoli cell (Flickinger and Fawcett, 1976; Fawcett, 1975; Russell and Clermont, 1976) and these structures are undoubtedly important in a variety of shape changes
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occuring during the spermatogenic cycle (Fawcett, 1975; Russell, 1977a, b; Burton et al., 1980; Suarez-Quian ef al., 1980). It should be emphasized that it would be naive to suggest that only cell structures involved by the treatments were microtubules. Recent research clearly shows that other elements, especially the system of microfilament, interact with microtubules (Griffith, 1980; Pollard, 1980; Wolosewick and Porter, 1976). Although the primary effects noted in this study are probably due to microtubular destabilization, the relative roles of microtubules and microfilaments in development and maintenance of the configuration of the Sertoli cell and its processes await further elucidation. The intent of the long-term studies was to assess the regenerative capabilities of the Sertoli cells and spermatogenic cells after shedding and elimination of the majority of the apical Sertoli cell and its attached germinal cells. First, one injected testis was processed at 12 hr post-injection to predict the severity of the response in the testicle of the opposite side. Once it was determined that a massive sloughing response was present on one side, the other testicle was allowed to remain in situ for about one cycle of the
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seminiferous epithelium (Clermont rt a/., 1959). At sacrifice, this testis was small and largely depleted of germ cells. The tubule showed numerous Sertoli cells which were relatively small but, nevertheless, much larger than many of those Sertoli cells from tubules of the 12 hr testis which had undergone sloughing of their apical components to the level of the blood-testis barrier. The extent of the ability of these cells to regenerate their apical cytoplasmic processes remains in doubt since morphometric studies were not performed; but our subjective observations suggest that the Sertoli cells appear to have considerable ability to regenerate apical cytoplasm and repopulate their processes with microtubules. The necessity for the presence of a population of germ cells for the Sertoli cell to assume its typical configuration is underscored. Acknowledgements The use of the Springfield S.M. Electron Microscope Facility is acknowledged. Mr R. Venezia provided excellent technical assistance in this project supported by a special in-house (SIU) Research, Development and Administration Grant.
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TILNEY, L. G. and PORTER, K. R., 1965. Studies on the microtubules of Heliozoa. 1. Fine structure in Actinosphaerium with particular reference to enamel rod structure. Protop/asma, 50, 317-344. VITALE, R., FAWCETT, D. W. and DYM, M. 1973. The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment. Anar. Rec., 176, 333-344. WOLOSEWICK, J. J. and BRYAN, J. H. D. 1977. Ultrastructural characterization of the manchette microtubules in the seminiferous epithelium of the mouse. Am. J. Amt., 150, 301-332. WOLOSEWICK, J. J. and PORTER, R. R. 1976. Stereo high-voltage electron microscopy of whole cells of the human diploid line. Am. J. Anar. 147, 303-324. WONG, V. and RUSSELL, L. D. 1981. Three-dimensional reconstruction of a Stage V rat Sertoli cell: methodological considerations. Amt. Rec.. 199, 281a.