Distribution of actin in isolated seminiferous epithelia and denuded tubule walls of the rat.код для вставкиСкачать
THE ANATOMICAL RECORD 213:63-71 (1985) Distribution of Actin in Isolated Seminiferous Epithelia and Denuded Tubule Walls of the Rat A.W. VOGL, L.J. SOUCY, AND G.J. LEW Department ofdnatomy, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada V6T 1 W5 ABSTRACT We have studied the distribution of actin, using NBD-phallacidin a s a probe, in isolated sheets of seminiferous epithelia and denuded tubule walls of the rat. Sheets of intact seminiferous epithelia were separated from tubule walls using EDTA in PBS. The isolated epithelia and denuded tubule walls were fixed, mounted on slides, made permeable with cold acetone ( -2OoC), and then treated with NBDphallacidin. Actin was observed in myoid cells, in ectoplasmic specializations of Sertoli cells, and in Sertoli cell regions adjacent to tubulobulbar processes of late spermatids. In myoid cells, filament bundles course in circular and longitudinal directions relative to the tubule wall. In Sertoli cells viewed at an angle perpendicular to the epithelial base, actin filaments in ectoplasmic specializations adjacent to junctional complexes circumscribe the bases of the cells. Filament bundles in ectoplasmic specializations adjacent to germ cells closely follow the contour of and are arranged parallel to the long axis of the developing acrosome. Sertoli cell regions adjacent to tubulobulbar processes of late spermatids stain intensely with NBD-phallacidin. Isolated seminiferous epithelia, combined with NBD-phallacidin as a probe for actin, provide a n ideal model system in which to study further the contractile properties of Sertoli cell ectoplasmic specializations and the possible involvement of these structures in events that occur during spermatogenesis. Sertoli cells are thought to be involved with many of the morphogenetic events that occur in the mammalian seminiferous epithelium during spermatogenesis (Fawcett, 1975; Russell, 1980a; Vogl et al., 1983). Among these events are the movement of spermatocytes through the blood testis barrier, the translocation of spermatids to the luminal surface of the tubule, and the release of spermatozoa from the epithelium. Although the underlying mechanisms of these events are unknown, the elaborate cytoskeletal network of Sertoli cells is likely involved (Fawcett, 1975; Means et al., 1980; Russell, 1980a; Vogl et al., 1983). Ectoplasmic specializations (Russell, 1977) are a particularly interesting group of Sertoli cell structures that are partly composed of cytoskeletal elements and that may be involved with some of the events described above. These structures occur adjacent to the basally situated junctions between Sertoli cells and in regions of adhesion to germ cells. They are characterized by a dense layer of filaments sandwiched between the plasma membrane and a cistern of endoplasmic reticulum (Brokelmann, 1963; Flickinger and Fawcett, 1967; Nicander, 1967; Dym and Fawcett, 1970). Because these regions contain actin (Toyama, 1976; Franke et al., 1978) and possess a magnesium-dependent ATPase activity (Gravis et al., 1976),they are generally thought to be contractile. Using techniques similar to those developed for studying intestinal epithelia (Bjerknes and Cheng, 19811, we have recently succeeded in isolating intact sheets of (i, 1985 ALAN R. LISS, INC seminiferous epithelia from the testes of ground squirrels (Vogl and Soucy, 1985). We are using this isolation procedure, together with NBD-phallacidin as a probe for filamentous actin (Barak et al., 19801, to study further the structure and function of Sertoli cell ectoplasmic specializations. This methodology has allowed us to visualize the three-dimensional arrangement of actin filament bundles in Sertoli cells and in myoid cells of seminiferous tubules. We initiated this study to determine the arrangement of actin filament bundles in seminiferous tubules of the rat. We chose t,he rat because it is used by most investigators as the model system in which to study mammalian spermatogenesis. MATERIALS AND METHODS Three adult male Sprague-Dawley rats (392, 396, 375) were used for these studies. Seminiferous epithelia and denuded tubule walls were obtained in the following manner. The testes were excised from animals anesthetized with sodium pentobarbitone administered intraperitoneally and perfused, via the spermatic artery on the posterior aspect of each testis, with phosphate-buffered saline (PBS) (150 mM NaC1; 5 mM KC1; 0.8 mM KHzPO,; 3.2 mM NazHPO,; Received August 27, 1984; accepted March 28, 1985 64 A.W. VOGL, L.J. SOUCY, AND G.J. LEW adjusted to pH 7.3 with 0.1 N NaOH) containing 20 mM EDTA (ethylenediamine-tetraacetic acid). After 5 minutes, perfusion was stopped and the testes were transferred to a petri dish containing 5 mM EDTA in PBS. The organs were then decapsulated and the seminiferous tubule masses cut, using two scalpels in a scissorlike fashion, into small segments. During this procedure, seminiferous tubules separated from interstitial tissue, and sheets of seminiferous epithelium separated from tubule walls. Identification of the various components was faciliated by using a Zeiss Stereomicroscope SR fitted with a darkfield condensor. Interstitial tissue was teased away from tubular elements, and the latter, containing epithelial sheets, denuded tubule walls, and intact tubules, were collected with a polyethylene pipette and transferred to a 15-ml centrifuge tube. Total time from decapsulating a testis to completing the harvesting of tubular elements was 10 minutes. All solutions used in the protocol described above were at room temperature. Samples to be used for fluorescence microscopy were centrifuged at a low setting and the supernatants replaced with PBS containing 5 mM EDTA and 3.7% paraformaldehyde. After 10 minutes in fixative, the tissue was washed three times with PBS. To help visualize filaments associated with spermatogenic cells, some of the material was mechanically fragmented by aspiration with a syringe fitted with first an 18 gauge then a 25 gauge needle. Isolated epithelia, denuded tubule walls, and mechanically fragmented samples were mounted together on polylysine-covered slides, treated with cold acetone (-2O"C), and air-dried. Samples mounted on slides were rehydrated for 10 minutes with PBS, then exposed for 20 minutes, a t room temperature, to one of the following: 1)PBS (control for autofluorescence); 2) PBS + 1.65 x M NBD-phallacidin (fluorescent probe for filamentous actin); 3 ) PBS + 1.65 x lop6 M NBD-phallacidin + 1.04 x lo-* M phalloidin (competitive specificity control); 4) PBS + 1.04 x lop4 M phalloidin (control for phalloidin in treatment 3). The slides were washed twice with PBS then mounted with 1:l (by volume) glycero1:PBS containing 0.02% sodium azide. Fluorescence was recorded on Tri-X film using Zeiss Photomicroscope I11 fitted with filters used for detecting fluorescein. Spermatids were staged using criteria established by Leblond and Clermont (1952). Tissue for electron microscopy was processed as follows. A sample of tubular elements containing a mixture of epithelial sheets, denuded tubule walls, and intact tubules was centrifuged a t a low setting and the supernatant replaced with a fixative containing 1.5% glutaraldehyde, 1.5% paraformaldehyde, and 0.1 M sodium cacodylate (pH 7.3). After 2 hours, the sample was washed with buffer then some of the material was aspirated through first an 18 gauge then a 25 gauge needle. All tissue was postfixed on ice for 1 hour with 1%Os04 in 0.1 M sodium cacodylate (pH 7.3).The material was processed further using standard techniques for electron microscopy. Thick sections (1 pm) were photographed with a Zeiss Photomicroscope 111. Thin sections were studied with a Phillips 300 operated a t 60 kV. of intact seminiferous tubules, sheets of seminiferous epithelium, and denuded tubule walls. These different elements were visible with darkfield optics during the isolation treatments and their presence was confirmed in thick sections of fixed material (Figs. 1,2). Two major features were evident in tissue viewed with the electron microscope. First, the isolated sheets of seminiferous epithelium lacked a basal lamina (data not shown). Second, ectoplasmic specializations adjacent to germ cells and Sertoli cell junctional complexes remained intact. In fact, ectoplasmic specializations of the Sertoli cell often remained attached to spermatids that had been mechanically dissociated from the epithelium (Figs. 3,4). Using NBD-phallacidin as a probe for actin, we observed strong fluorescence in myoid cells and in Sertoli cells. In Sertoli cells, ectoplasmic specializations were labeled, as were regions adjacent to tubulobulbar complexes. Although the fluorescence emitted from labeled actin in myoid cells was most clearly evident in denuded tubule walls, such as the one shown in Figure 5, it was also visible in intact seminiferous tubules (not shown). In the latter structures, the fluorescence pattern of labeled actin in myoid cells was visualized simultaneously with that in Sertoli cell junctional complexes. Within each flat and somewhat polygonally shaped myoid cell, linear tracts of actin were observed extending across the cell from one border to another as shown in Figure 5 . These tracts were oriented predominantly in two directions, with those adjacent to one cell surface organized in a circular manner relative to the seminiferous tubule wall and those adjacent to the other surface arranged longitudinally. The pattern of actin we observed in Sertoli cells was very different from that in myoid cells. A strong fluorescence was emitted by actin in ectoplasmic specializations of junctional complexes and appeared to outline the base of each Sertoli cell. When an epithelial sheet was viewed perpendicular to the epithelial base, as in Figure 6, a honeycomb pattern was observed. By adjusting the plane of focus, or by looking a t the edges of an epithelial sheet where the elongate Sertoli cells were attached by their lateral surfaces to the slide (Fig. 71, the distribution of NBD-phallacidin in more apical epithelial regions could be observed. In apical regions, fluorescence was associated almost exclusively with developing spermatids. Although certainly evident in intact epithelial sheets, the fluorescence emitted from actin in these regions was more easily studied in fragmented epithelia. Unlike the fluorescence in regions adjacent to Sertoli cell tight junctions, the pattern of fluorescence in apical epithelial regions associated with spermatids was different a t different stages of spermiogenesis. Because 1)we had demonstrated that ectoplasmic specializations of Sertoli cells often remained attached to spermatids that were mechanically dissociated from the epithelium, and 2) the fluorescence we observed appeared to closely follow the outer contours of the germ cells, we interpreted most of the fluorescence as coming from actin in ectoplasmic specializations of Sertoli cells and not from the germ cells themselves. The earliest stage of spermatid RESULTS with which staining was associated was approximately Using the isolation procedure described above, we were stage 8 (Fig. 8a, a'). At this stage, ectoplasmic specialiable to obtain testicular samples containing a mixture zations occurred adjacent to the region of the spermatid ACTIN IN SEMINIFEROUS TUBULES Fig. 1. Thick (1pm) section of isolated seminiferous epithelia (asterisk) and denuded tubule walls (arrows) obtained using techniques described in the text. Bar = 100 pm. x 347. Flg. 2. Thick (1 pm) section of an isolated sheet of seminiferous epithelium. Notice that a tubule wall is absent. A group of three spermatogonia is indicated by the arrow. Bar = 150 pm. x 198. Fig. 3. Electron micrograph of a spermatid that has separated from the seminiferous epithelium. An ectoplasmic specialization (arrow- 65 heads) of a Sertoli cell is attached to the region juxtaposed to the acrosome. Bar = 2.5 pm. ~ 9 , 7 8 9 . Fig. 4. A magnified view of the region indicated by the rectangle in Figure 3. Filament bundles within the attached Sertoli cell ectoplasmic specialization are indicated by the arrowhead. Also indicated are 1)the nuclear envelope of the spermatid, 2) inner acrosomal membrane, 3) outer acrosomal membrane, 4) plasma membrane of the spermatid, 5) plasma membrane of the Sertoli cell, and 6) “outer” membrane of a Sertoli cell cistern of endoplasmic reticulum. Bar = 0.50 pm. ~ 6 4 , 0 0 0 . 66 A.W. VOGL, L.J. SOUCY, AND G.J. LEW Fig. 5. Distribution of NBD-phallacidin in myoid cells of a denuded seminiferous tubule wall. A single myoid cell is indicated by the arrows. The fluorescence occurs as linear tracts that course across the cells in predominantly two directions. Bar = 50 pm. x590. Fig. 6. Actin distribution, as indicated by NBD-phallacidin, at the base of an isolated sheet of seminiferous epithelium. The base of a single Sertoli cell is indicated by the arrows. The fluorescence corre- sponds to the position of ectoplasmic specializations adjacent to junctions between Sertoli cells. Bar = 40 pm. X890. Fig. 7. Actin distribution predominantly in Sertoli cell ectoplasmic specializations adjacent to spermatids. Shown in this figure are clusters of spermatids that occur in apical recesses of Sertoli cells. Notice that the spermatid heads are clearly marked by the fluorescence emitted by ectoplasmic specializations in the adjacent Sertoli cells. Bar = 40 pm. X730. 67 ACTIN IN SEMINIFEROUS TUBULES Fig. 8. Shown here is the distribution of actin, a s indicated by the probe NBD-phallacidin, i n ectoplasmic specializations adjacent to sequential stages of germ cells. The germ cells present in these micrographs were mechanically separated, together with adjacent Sertoli cell regions, from epithelia isolated a s described in the text. A, acrosome; N, nucleus; RC, residual cytoplasm. Shown in panels a (fluorescence) and a ’ (phase) is a spermatid a t approximately stage 8 of spermiogenesis. Bundles of actin filaments are obvious in the discshaped ectoplasmic specialization t h a t lies adjacent to the acrosome. In panels h and b’ (stage 10 spermatid) and c and c’ (stage 11spermatid) the fluorescence pattern closely follow the contours of the acrosomes. Filament bundles are aligned parallel to the long axis of the acrosomes. In panels d and d’ (stage 15-17 spermatids) and e and e’ (stage 19 spermatids) ectoplasmic specializations outline the shapc of the underlying spermatid heads. Also shown in panel e is a n intense fluorescence emitted from clusters of columnar structures present along the concave surface of the spermatids. In the region marked by the arrows, notice t h a t the columnar units occur in two rows. We believe t h a t signals such a s these are from the filament~richSertoli cell regions surrounding tubulobulbar processes of the germ cells. Bar = 10 pm. ~ 1 , 5 4 0 . plasma membrane overlying the developing acrosome and consisted of linear tracts of fluorescence that together formed a disc-shaped unit. At later stages, the fluorescence closely followed the contours of the developing acrosome (Fig. 8b,b’,c,c’)and eventually the entire spermatid head (Fig. 8d,d’,e,e‘). At these later stages of spermiogenesis, linear tracts of actin were observed that generally aligned parallel to the long axis of the acrosome andor spermatid head (Figs. 8, 9). In Sertoli cell cytoplasm adjacent to stage 19 spermatids, linear tracts of fluorescence occasionally were observed extending from the tip of the spermatid head to more basal head regions, as shown in Figure 10a. The most intense fluorescence we observed was not emitted by actin in myoid cells or in ectoplasmic specializations of Sertoli cells, but rather by actin in regions adjacent to the concave surface of stage 19 spermatids (Fig. 8e, 9a,b). Often the fluorescence pattern appeared to consist of two rows of columnar units all aligned perpendicular to the rim of the spermatid head. This is particularly evident in Figure 8e and is less visible in Figures 9a and b. This region of Sertoli cells is closely associated with the tubulobulbar processes of late-stage spermatids. No specific fluorescence was observed in any of the control slides (Fig. 10). DISCUSSION In seminiferous tubules of the rat, actin concentrations, as visualized with NBD-phallacidin, occur in three major locations: 1)myoid cells; 2) ectoplasmic specializations of Sertoli cells; 3) regions of the Sertoli cell around tubulobulbar processes of spermatids. In myoid cells, actin filament bundles course predominantly in two directs. Those lying adjacent to one flat surface of the cells course in a circular direction relative 68 A.W. VOGL, L.J. SOUCY, AND G.J. LEW Fig. 9. Fluorescence (a,b) and phase (a’,b’)micrographs of stage 19 spermatids, and adjacent Sertoli cell regions, labeled with NBD-phallacidin. The arrows indicate the orientations of actin filament bundles in ectoplasmic specializations. An intense fluorescence is evident ad jacent to the concave surface of the spermatid heads. That this signal is composed of columnar units, similar to those shown in Figure 8e, is indicated in regions marked by the arrowheads. Bar = 5 pm. X4,OOO. to the tubule wall while those on the other course longitudinally. This arrangment probably enables the single layer of myoid cells to generate contractile forces in more than one direction. That myoid cells contain actin and are contractile has been appreciated for some time (Clermont, 1958; Toyama, 1977). However, the three dimensional arrangement of filaments within these cells has not been previously been described. Our observations of the arrangement of actin filaments in Sertoli cell ectoplasmic specializations are consistent with the actin patterns visible in published electron micrographs of these regions. At basally situated junctions between Sertoli cells, filaments lie sandwiched between the plasma membrane and a cistern of endoplasmic reticulum (Brokelmann, 1963; Dym and Fawcett, 1970; Flickinger and Fawcett, 1967; Fawcett, 1975; Nicander, 1967). In the present study, we have shown that actin filaments at these junctional sites circumscribe the bases of Sertoli cells. When intact epithe- lial sheets are viewed from a n angle perpendicular to the epithelial base, and numerous Sertoli cell junctional complexes are seen simultaneously, a “honeycomb” arrangement is evident. These results are consistent with predictions based on three-dimensional reconstructions from electron micrographs of Sertoli cell junctional complexes (Weber et al., 1983). In ectoplasmic specializations associated with germ cells, actin filaments occur in linear bundles that are generally arranged parallel to the long axis and conform to the shape of the developing acrosome. Although Russell et al. (1980) have demonstrated in electron micrographs that ectoplasmic specializations occur adjacent to spermatogenic cells a t stages as early as spermatocytes and round spermatids, in the present study, using fluorescence, we observed these structures as distinct entities only in association with approximately stage 8 and later spermatids. It is unclear why there is this discrepancy between the two sets of data. One possible ACTIN IN SEMINIFEROUS TUBULES 69 Fig. 10. Control series for NED-phallacidin staining. Shown in panels a (fluorescence) and a‘ (phase) is a spermatid stained with NBD-phallacidin. Actin bundles in the ectoplasmic specialization are labeled as are filaments within the concavity of the spermatid head. Also notice the linear signal that courses across the Sertoli cell apical process from the tip of the spermatid head to more caudal regions of the head. In panels b and b‘, c and c’, and d and d’ are spermatids treated with 1) NBD-phallacidinin the presence of phalloidin, 2) phalloidin alone, and 3) buffer alone, respectively. Bar = 10 pm. X 2 , l O O . reason is that specializations associated with spermatocytes and round spermatids may be remnants of Sertoli cell junctional complexes and hence not recognized as distinct entities by fluorescence. When they first appear, ectoplasmic specializations have a disc shape and are composed of linear bundles of filaments. As spermatogenesis continues the bundles become less distinct, but are still visible, and the entire structure undergoes shape changes that coincide with the shape changes in the developing acrosome. Bundles of filaments that we detected in those cytoplasmic regions of Sertoli cells associated with stage 19 spermatids generally correspond to the orientation of filament bundles visible in electron micrographs published, for example, by Lalli and Clermont (1981); that is, the bundles are generally oriented along the long axis of the spermatid head and occasionally are visible in Sertoli cell regions not directly apposed to the germ cell. Although we have interpreted the fluorescence associated with germ cells as originating from actin in attached Sertoli cell ectoplasmic specializations, we cannot rule out the possibility that some of the signal, particularly that associated with stage 10 and 11 spermatids, may actually have been generated by filamentous actin within the germ cells themselves. However, three pieces of information are consistent with our conclusion that most of the observed fluorescence was due to actin in ectoplasmic specializations. First, ultrastructural data indicate that ectoplasmic specializations often remain attached to germ cells that are mechanically dissociated from the epithelium (this study; Romrell and Ross, 1979). Second, most of the fluorescence associated with germ cells appears to originate from a region external to the germ cell and not from the subacrosomal space-a location where filamentous actin is known to occur (Campanella et al., 1979). Third, the fluorescence pattern is consistent with the arrangement of actin filament bun- dles in electron micrographs of ectoplasmic specializations. The fluorescence pattern originating from actin in germ cells that are devoid of Sertoli cell fragments has yet to be determined. The function of ectoplasmic specializations is not known. The close relationship of these structures to Sertoli cell junctions and to regions of adhesion to germ cells indicates that they may be involved with intercellular attachment and hence with spermiation (Gravis, 1978a, 1979, 1980; Romrell and Ross, 1979; Ross, 1976, 1977; Ross and Dobler, 19751, as well as with the movement of germ cells through the blood testis barrier (Dym and Fawcett, 1970). Ectoplasmic specializations associated with germ cells have also been suggested to actively “grip” elongate spermatids (Franke et al., 1978; Toyama, 1976)and to “rigidify” Sertoli cell apical crypts in which spermatids mature (Russell, 1977, 1980a). Although ectoplasmic specializations are generally considered contractile (Gravis, 1978b, 1979; Toyama, 1976) we have been unable to demonstrate myosin in these structures or to induce their contraction in ground squirrel Sertoli cells (Vogl and Soucy, 1985). Our observation that ectoplasmic specializations adjacent to germ cells generally conform to the contour of the developing acrosome is consistent with, but does not prove, the hypothesis that the structures may facilitate the development of acrosome shape (Fawcett, 1979) or stabilize junctional sites in some way (Vogl and Soucy, 1985). It is also interesting that filament bundles are generally oriented parallel to the long axis of the spermatid head and not in a circular fashion as one might expect if ectoplasmic specializations “grip” the germ cells using contractile force. During the late stages of germ cell differentiation in the rat, two rows of tubular projections extend from the concave surface of the sickle-shaped spermatid heads into the adjacent Sertoli cell. These projections, together 70 A.W. VOGL, L.J. SOUCY, AND G.J. LEW with modified regions of the Sertoli cell, are termed tubulobulbar complexes (Russell and Clermont, 1976). These complexes consist, in part, of a filamentous network in the Sertoli cell. This filamentous network may develop from a rearrangement of actin in ectoplasmic specializations. This argument is supported by the observation that tubulobulbar processes appear in regions associated with ectoplasmic specializations (Russell and Clermont, 1976). The function of tubulobulbar complexes is not known. The most popular working hypotheses are that they assist in attaching spermatids to the epithelium (Russell and Clermont, 1976; Russell, 1979a) or facilitate the removal of excess cytoplasm from maturing germ cells (Russell, 1979b, 1980b, see review by Russell, 1984). It has also been suggested that they transfer a chemical signal to Sertoli cells that initiates sperm release (Gravis, 1980). Our results indicate that filament networks in regions of the Sertoli cell adjacent to tubulobulbar processes contain a n abundance of actin. Actin in these regions may be contractile, or may be purely skeletal without being contractile. Although the functional significance of these possibilities has yet to be demonstrated, the actin networks may play a role in anchoring the tubulobulbar processes to the Sertoli cell, facilitating the development of spermatid head shape, or internalizing germ cell cytoplasm. In summary, we have been able to visualize the threedimensional arrangement of actin filament bundles in myoid cells and in ectoplasmic specializations of Sertoli cells in the rat. We have also presented evidence indicating that the filament networks in Sertoli cell regions surrounding tubulobulbar processes of spermatids contain actin. Isolated sheets of seminiferous epithelia may provide a n ideal system in which to study further the involvement of Sertoli cell cytoskeletal elements in events that occur during spermatogenesis. ACKNOWLEDGMENTS We would like to thank Marilyn Stuart for typing the manuscript and Bryon Grove for many helpful criticisms. This work was supported by BCHCRF grant #67 (83-1)and MRC grant #MA-8020 to A.W. Vogl. LITERATURE CITED Barak, L.S., R.R. Yocum, E.A. Nothnagel, and W.W. Webb (1980)Fluorescencestaining of the actin cytoskeleton in living cells with 7-nitrobem-2- oxa-l,3-diazole-phallacidin. Proc. Natl. Acad. Sci. USA, 77:980-984. Bjerknes, M., and H. Cheng (1981) Methods for the isolation of intact epithelium from the mouse intestine. Anat. Rec., 199565-574. Brokelmann, J. (1963) Fine structure of germ cells and Sertoli cells during the cycle of the seminiferous epithelium in the rat. Z. Zellforsch. Microsk. Anat., 592320-850. Campanella, C., G. Gabbiani, B. Baccetti, A.G. Burrini, and V. Pallini (1979) Actin and myosin in the vertebrate acrosomal region. J. Submicrosc. Cytol., 1153-71. Clermont, Y. 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Osborn (1978) Location of actin i n microfilament bundles associated with the junctional specializations between Sertoli cells and spermatids. Biol. Cell. 31r7-14. Gravis, C.J., R.D. Yates, and I-Li Chen (1976) Light and electron microscopic localization of ATPase in normal and degenerating testes of Syrian hamsters. Am. J. Anat., 147;419-432. Gravis, C.J. (1978a) Inhibition of spermiation i n the Syrian hamster using dibutyryl cyclic-AMP,Cell Tissue Res., 192:241-248. Gravis, C.J. (197813) A scanning electron microscopic study of the Sertoli cell and spermiation in the Syrian hamster. Am. J. Anat., 151:21-38. Gravis, C.J. (1979) Interrelationships between Sertoli cells and germ cells in the Syrian hamster. Z. Mikrosk. Anat. Forsch. (Leipz.), 93:321-342. Gravis, C.J. (1980) Ultrastructural observations on spermatozoa retained within the seminiferous epithelium after treatment with dibutyryl cyclic-AMP. Tissue Cell, 12309-322. Lalli, M., and Y. 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Cell Tissue Res., 177:221-226. Vogl, A.W., Y.C. Lin, M. Dym, and D.W. Fawcett (1983) Sertoli cells of 71 ACTIN IN SEMINIFEROUS TUBULES t h e golden-mantled ground squirrel (Sperrnophilus lateralis): A model system for t h e study of shape change. Am. J. Anat., 168.8398. Vogl, A.W., and L. Soucy (1985) Arrangment and possible function of actin filament bundles i n ectoplasmic specializations of ground squirrel Sertoli cells. J. Cell Biol., 100: 814-825. Weber, J.E., L.D. Russell, V. Wong, and R.N Peterson (1983) Threedimensional reconstruction of a r a t stage V Sertoli cell: 11. Morphometry of Sertoli-Sertoli and Sertoli-Germ-cell relationships. Am. J. Anat.. 167:163-179. NOTE ADDED IN PROOF Suarez-Quian and Dym (1984, Annals of the New York Academy of Sciences, Vol. 438, pp. 476-480) recently reported on the distribution of actin, as indicated by NBD-phallacidin, in fixed and frozen sections of rat testis. Our results confirm and extend their original findings. These authors also reported that they could not demonstrate myosin in Sertoli cell junctional complexes, and suggested that microfilaments a t these sites stabilize the blood-testis barrier.