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We have investigated, using indirect immunofluorescence techniques, the possibility that vinculin is a component of Sertoli cell ectoplasmic specializations. Affinity-purified polyclonal antibodies produced against human platelet vinculin were used to probe fixed frozen sections of rat testis. Specific fluorescence occurs in Sertoli cell regions adjacent to spermatids and to basally situated junctional complexes, sites at which ectoplasmic specializations are known to occur. Staining also occurs in Sertoli cell regions associated with tubulobulbar complexes. The antibody also labels focal contacts in cultured human dermal fibroblasts, apical junctional sites of rat epididymal epithelium, and dense plaques of smooth muscle. Our results are consistent with the prediction that vinculin is likely a component of ectoplasmic specializations and are also consistent with the hypothesis that these structures are a form of actin-associated adhesion complex.

Study of Sertoli cells of the ground squirrel provides a unique opportunity to examine cell structure and function. The cells are large, have an elaborate cytoskeleton, and undergo dramatic changes in organization during spermatogenesis. Microtubules (MTs) are prominent elements of the cytoskeleton and appear to be associated structurally with many of the events that occur during sperm production. To investigate the function of MTs, animals were injected subcutaneously with colchicine, and their seminiferous epithelia examined by light and electron microscopy. Some animals were injected with 30--80 mg of the drug per kg body weight and sacrificed 3 to 5 hr later. Others were given 0.3 mg/kg/day for 6 days and processed on day 7. Virtually no MTs were seen in Sertoli cells after short-term treatments, and their numbers were greatly reduced after the long-term injections. Intermediate filaments were very evident throughout the cytoplasm of treated cells, particularly in the short-term studies. Moreover, a close association of some of these filaments with centrioles was observed. In all cases, elongate spermatids which normally move apically did not do so. Indeed, some spermatids appear to have been pulled to a basal position after having moved apically prior to treatment. Also, smooth endoplasmic reticulum (SER) accumulated basally in the Sertoli cell, unlike controls, and the acrosomes of late spermatids developed abnormally or did not complete their shape changes. Cell junctions appeared normal and sperm release was observed. In conclusion, our data suggest that Sertoli cell MTs are necessary for the normal development and translocation of spermatids in the seminiferous epithelium and are involved with positional changes in Sertoli cell SER. They do not appear essential for the maintenance of cell junctions.

In this paper we document retial supply of the spinal cord and describe the arterial vascular pattern of the brain in the whale family Monodontidae. Observations are based on gross dissections of four brains, two each of Monodon monoceros and Delphinapterus leucas, and one spinal cord from M. monoceros. Vessels of the spinal cord arise from extradural retia in the neural canal. Arteries originating from the retia penetrate the dura between successive spinal roots (mainly ventral) and not in association with them, unlike radicular arteries of other mammals. Also, these vessels are uniformly distributed and contribute equally to a plexus surrounding the cord. An A. radicularis magna is not present, and neither are distinct anterior or posterior spinal arteries. Circulation to the brain is effected by two pairs of arteries originating from intracranial retia. The rostral pair supplies most of the forebrain (prosencephalon), whereas the more caudal pair supplies mainly the midbrain (mesencephalon) and hindbrain (rhombencephalon). The circulatory pattern is characterized by 1) complete independence of anterior cerebral arteries (no anastomoses); 2) extensive cortical supply by the anterior choroidal arteries; 3) absence of subdural communicating vessels between rostral and caudal trunks; 4) union of caudal trunks to form a small basilar artery; and 5) absence of vertebral arteries and hence of a vertebral basilar system. There are some obvious differences between subdural arteries in the Monodontidae and those in other mammals; however, their general patterns of distribution are similar, and we suggest that most of the vessels, at least in the cranium, are homologous.

We have studied the ultrastructure of Sertoli-cell processes that extend into developing germ cells of the ground squirrel (Spermophilus lateralis). In other mammals it is speculated that these processes anchor germ cells to the seminiferous epithelium and transfer materials between Sertoli and germ cells. In the ground squirrel, Sertoli-cell projections first appear in round spermatids and consist of regions containing numerous mitochondria and intermediate filaments together with areas composed mainly of a fine filamentous matrix. Also present are what may be desmosomelike junctions with adjacent germ cells. During spermatogenesis, numerous changes in the penetrating processes and their internal composition occur. Especially significant are those occurring during the movement of residual cytoplasm basally over spermatid heads: some Sertoli-cell processes contain microtubules, mitochondria, and vesicular elements, but also present are regions that lack organelles and appear simply as thin lamellae of cytoplasm that line cavernous invaginations of the germ cell. Coated vesicles and pits are present in processes and adjacent germ-cell regions at all stages of spermatogenesis. Our observations are consistent with the suggestions that Sertoli-cell processes have an attachment function and that they also may facilitate the movement of residual cytoplasm into the epithelium. Further, they indicate that these structures might be involved with receptor-mediated edocytosis.

We have studied changes in myoepithelial cell size and shape during different stages of mouse mammary gland differentiation by using the fluorescent probe for actin NBD-phallacidin. Pieces of mammary tissue were fixed, mounted on slides, permeabilized with cold acetone (-20 degrees C), and then treated with nitrobenzoxadiazole-phallacidin. Myoepithelial cells lining ducts of glands at all stages of development are spindle-shaped structures oriented parallel to the long axis of the duct at the base of the luminal epithelium. In virgin animals, myoepithelial cells also occur as linear tracts oriented parallel to the long axis of small projections along the sides of ducts and terminal end buds. In early pregnancy, small stellate-shaped cells begin to appear around presumptive secretory units. By late pregnancy, larger star-shaped units of intense fluorescence appear at the base of alveoli. During lactation, both cell bodies and cell processes further enlarge as these interlacing stellate-shaped cells encompass the expanded alveoli. In regressing glands, cell size decreases and the processes appear to retract. Although alveoli are virtually absent in the multipartate resting gland, myoepithelial cells remain around lateral buds of ducts. These myoepithelial cells have two distinct shapes: small star-shaped cells capping the buds and spindle-shaped cells oriented parallel to the long axis of the buds. A comparison of myoepithelial cell shape in virgin mice and nulliparous women indicates a more developed cell in the human gland at this stage of development. Intact segments of mammary gland combined with NBD-phallacidin as a probe for actin provide an ideal system for future studies of the control of myoepithelial cell size and shape and their influence on cell functions.

Sertoli cells of the ground squirrel (Spermophilus lateralis), a seasonal breeder, were examined by light and electron microscopy and their structure, particularly the organization of the cytoskeleton, was related to events that occur in the seminiferous epithelium during spermatogenesis. Among the events considered and described are the apical movement of elongate spermatids, withdrawal of residual cytoplasm from germ cells, transport of smooth endoplasmic reticulum (SER) between the base and apex of the Sertoli cells, and sperm release. These events are dramatically evident in this species because the seminiferous epithelium is thin, i.e., there are few germ cells, and both the germ cells and Sertoli cells are large. Sertoli cells of the ground squirrel have a remarkably well developed cytoskeleton. Microfilaments occur throughout the cell but are most evident in ectoplasmic specializations associated with junctions. Intermediate filaments occur around the nucleus, as a layer at the base of the cell, and adjacent to desmosome-like junctions with germ cells. Intermediate filaments, together with microtubules, are also abundant in regions of the cell involved with the transport of SER, in cytoplasm associated with elongate spermatids, and in processes that extend into the residual cytoplasm of germ cells. Our observations of ultrastructure are consistent with the hypothesis that Sertoli cell microtubules are involved with the movement of germ cells within the seminiferous epithelium, and further implicate these structures as possibly playing a role in the retraction of residual cytoplasm from germ cells and the intracellular transport of SER. The abundance and organization of intermediate filaments suggest that these cytoskeletal elements may also be involved with events that occur during spermatogenesis.

I have studied the distribution of microtubules in rat Sertoli cells during spermatogenesis. The distribution of microtubules was determined by indirect immunofluorescence in fragments of seminiferous epithelium which were mechanically dissociated from perfusion-fixed testes. The presence of microtubules in regions indicated by immunofluorescence was confirmed with electron microscopy. Microtubules are not evident in Sertoli cell regions surrounding early spermatogenic cells, but are abundant in cytoplasm adjacent to apical crypts containing elongate spermatids. In regions surrounding crypts, microtubules are uniformly aligned parallel to the long axis of Sertoli cells. Microtubules in these more central, or columnar, regions of the cells extend from supranuclear levels to the apex of the epithelium. In apical processes surrounding late spermatids, microtubules conform to the contours of the hook-shaped spermatid heads. These microtubules appear continuous with those in the columnar regions of the Sertoli cells. Changes in microtubule distribution observed in the rat are generally similar to, but less elaborate than, those reported previously in the squirrel. These changes may be characteristic of mammalian spermatogenesis in general.

We have studied the distribution of actin, using NBD-phallacidin as 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 (-20 degrees C), and then treated with NBD-phallacidin. 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 an 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.

Cytochalasin D (CD) was used to perturb actin filaments of the Sertoli ectoplasmic specialization (ES)--a cytoskeletal complex of the Sertoli cell related to spermatids. CD (500 microM for 6 h) produced a loss of 88% of the ES facing the head region of early (Step 8) elongating spermatids as compared to vehicle (dimethylsulfoxide:saline) controls. Nitrobenzoxadiazole-phallacidin staining of F-actin revealed a CD-related loss of uniform fluorescence over the head of elongated spermatids. To examine for a possible relationship between the presence of actin and cell attachment at ES sites, hypertonic fixatives were introduced to provoke cell shrinkage and stress ES-associated junctions. After osmotic stress, cell-to-cell adhesion at ES sites remained intact in vehicle-treated animals. CD treatment caused Sertoli cells to separate from elongating spermatids at sites where ES had been lost from the Sertoli cell surface. It is suggested that actin of the ES plays a role in cell-to-cell interaction analogous to its possible role at the Sertoli cell barrier. In CD-treated animals, structures resembling tubulobulbar complexes frequently developed at sites where ES was lost, suggesting that the loss of ES has a facilitatory role in tubulobulbar complex formation. It is hypothesized that tubulobulbar complexes are devices that rid the cells of ES-associated junctional links to effect dissociation of the spermatid from the Sertoli cell during spermiation. Spermatids at Step 8 of development are known to become oriented with their acrosomes facing the base of the Sertoli cell. After CD treatment, a 5.8-fold increase in malorientation of Step 8 spermatids was noted. A role for the ES cytoskeletal complex in orienting the spermatid acrosome toward the basal aspect of the Sertoli cell is also suggested.

Two types of filaments were observed within the subacrosomal space of rat spermatids. The first of these types was characterized as actin by demonstration of actin filament affinity for myosin S-1 subfragments. Actin filaments were noted in the subacrosomal space shortly after the acrosomal sac made contact with the nucleus. As the acrosome increased its surface area contact with the spermatid nucleus, the number of layers of subacrosomal filaments increased. Pre-treatment with detergent, which in addition to permeablizing cells to allow entry of S-1, also caused the acrosome to vesiculate and the subacrosomal space to widen. In such preparations filaments were more easily visualized and appeared to extend between the nuclear and acrosomal membranes, indicating, but not proving, attachment to these membranes. During spermatid clongation, the number of actin filaments in the subacrosomal space increased greatly, especially over the dorsal convex region of the spermatid head. The polarity of the majority of filaments was not ascertainable since filaments were tightly packed within the narrow subacrosomal space. In late spermiogenesis (steps 18 and 19), actin filaments were no longer detected within the subacrosomal space. A second and much thicker type of filamentous structure was observed in the subacrosomal space of spermatids at steps 14-17 of spermiogenesis. About 14 nm in diameter (10-15 nm measurement range depending on fixation protocol utilized), these filaments did not decorate with myosin S-1 subfragments and were found in subacrosomal regions not containing actin. Fourteen nanometer filaments were seen in parallel array along the ventral folded portion of the nuclear membrane and extended partially around the nucleus. Like actin filaments. 14 nm filaments were not seen in the subacrosomal space during late spermiogenesis.

We have investigated the arrangement and function of actin filament bundles in Sertoli cell ectoplasmic specializations found adjacent to junctional networks and in areas of adhesion to spermatogenic cells. Tissue was collected, from ground squirrel (Spermophilus spp.) testes, in three ways: seminiferous tubules were fragmented mechanically; segments of intact epithelium and denuded tubule walls were isolated by using EDTA in a phosphate-buffered salt solution; and isolated epithelia and denuded tubule walls were extracted in glycerol. To determine the arrangement of actin bundles, the tissue was fixed, mounted on slides, treated with cold acetone (-20 degrees C), and then exposed to nitrobenzoxadiazole-phallacidin. Myosin was localized using immunofluorescence. To investigate the hypothesis that ectoplasmic specializations are contractile, glycerinated models were exposed to exogenous ATP and Ca++; then contraction was assessed qualitatively by using nitrobenzoxadiazole-phallacidin as a marker. Actin bundles in ectoplasmic specializations adjacent to junctional networks circumscribe the bases of Sertoli cells. When intact epithelia are viewed from an angle perpendicular to the epithelial base, honeycomb staining patterns are observed. Filament bundles in Sertoli cell regions adjacent to spermatogenic cells dramatically change organization during spermatogenesis. Initially, the bundles circle the region of contact between the developing acrosome and nucleus. They then expand to cover the entire head. As the spermatid flattens, filaments on one side of the now saucer-shaped head orient themselves parallel to the germ cell axis while those on the other align perpendicularly to it. Before sperm release, all filaments course parallel to the rim of the head. Contrary to the results we obtained with myoid cells, we could not convincingly demonstrate myosin in ectoplasmic specializations or induce contraction of glycerinated models. Our data are consistent with the hypothesis that actin in ectoplasmic specializations of Sertoli cells may be more skeletal than contractile.

We have studied the comparative anatomy of arterial plexuses (retia mirabilia) related to supply of the central nervous system in two closely related species of toothed whales - narwhal (Monodon monoceros) and beluga Delphinaterus leucas). In both species, retia originate from major vessels in the neck, thorax, and lumbar regions, then extend into the neural canal and cranium to supply the spinal cord and brain. The system generally consists of arteries embedded in á matrix of fatty connective tissue. Constituent vessels are only occasionally reated to veins or venous sinuses. Though retial anatomy is similar in the two species, there are two related features that appear species specific: (1) amount of retia originating from the supreme intercostal arteries and (2) thoracic retial size. Both are larger in the narwhal, as are values for hematocrit and hemoglobin concentration, which, in this study, we use as indices of diving ability. Our data are consistent with the hypothesis that the retia are in some way linked to diving ability. The nature of this link is not known; however, we discuss our results in the context of the most popular hyotheses of retial function., (Copyright © 1982 Wiley-Liss, Inc.)

The effect of calcium on oxytocin-induced contraction of myoepithelial cells was visualized with NBD-phallacidin, a fluorescent stain for filamentous actin. In the absence of oxytocin, the cells appeared relaxed; long, branching processes radiated from the cell bodies. In the presence of 50 nM-oxytocin, myoepithelial cells contracted into smaller spoke-shaped bodies in which the arms were shorter and thicker. Electron microscopy confirmed the morphological differences between oxytocin-treated and untreated myoepithelium. To determine a role for extracellular calcium, tissue was incubated in EGTA, then exposed to oxytocin, with or without added calcium. Contraction occurred in the presence of oxytocin plus additional calcium but not in the absence of calcium. When the tissue was incubated with the calmodulin antagonist trifluoperazine (TFP) in calcium-containing medium, oxytocin did not induce myoepithelial cell contraction. These data support previous results obtained with a myosin light-chain phosphorylation assay implicating calcium and calmodulin in oxytocin-induced contraction. Furthermore, NBD-phallacidin visualization of myoepithelial cells demonstrates that the effect of calcium on contraction is physiologically significant.

We have studied the distribution of microtubules and intermediate filaments in mammalian Sertoli cells during spermatogenesis. The arrangement of microtubules was determined, by indirect immunofluorescence, in ground squirrel testes that were 1) fixed, mechanically fragmented, and attached to polylysine-coated slides, and 2) fixed, embedded in polyethylene glycol, and sectioned. Intermediate filament patterns were determined, also by indirect immunofluorescence, in sections of unfixed rat testis. Results from these studies were confirmed and extended using electron microscopy. Microtubules first become evident in lateral processes that embrace round spermatids. When spermatids elongate and become situated in apical crypts of Sertoli cells, the microtubules become oriented parallel to the long axis of Sertoli cells and surround the crypts. As spermatids mature and acquire a saucer shape, apical microtubules progressively concentrate in Sertoli cell regions adjacent to the acrosome and eventually form discrete C-shaped structures that disappear during spermiation. Intermediate filaments in rat Sertoli cells are centered around the nucleus. From perinuclear regions, filaments extend toward desmosome-like junctions with early spermatogenic cells and into the apical cytoplasm where they have a transient association with crypts containing elongate spermatids. Filaments amongst crypts are most evident in early stages of the spermatogenic cycle when apical crypts are situated deep within the epithelium. They become less evident and eventually disappear as spermatids assume a more apical position. Our fluorescence studies and ultrastructural analyses indicate that the association of intermediate filaments with crypts is specific to regions adjacent to the dorsal or convex aspect of spermatid heads. In these regions, approximately 8 to 12 uniformly aligned filaments are intimately associated with actin filaments in ectoplasmic specializations surrounding the crypts. We conclude that, like actin, the distribution of microtubules and intermediate filaments changes in Sertoli cells during spermatogenesis. The distribution of microtubules correlates with the irregular columnar shape of Sertoli cells. We suspect that the apically situated intermediate filaments may play a role in anchoring or positioning Sertoli cell crypts deep within the epithelium during the early stages of the spermatogenic cycle.

In this paper we describe the gross and microscopic anatomy of the internal carotid artery and demonstrate that this vessel does not directly supply blood to the brain, in the Monodontidae (order Cetacea). Our account is based on gross dissections and perfusion casts of the arterial vasculature in Delphinapterus leucas and Monodon monoceros and on histological material from the latter species. The internal carotid artery originates low in the neck and extends to the carotid rete at the base of the brain. The vessel tapers dramatically along its cervical course and changes from an artery elastic in nature to one more muscular. A single large cervical branch occurs in D. leucas and supplies cerebrally related retia in this region and prevertebral muscles. No cervical branches occur in M. monoceros. In otic regions, the internal carotid artery is small and muscular. A lumen is present; however, a split internal and external elastic lamella and a thickened subendothelial layer are evident. Though patent in the neck and ear, the vessel appears occluded within the carotid canal. At this level, the vessel is characterized by absence of a lumen and by fragmented elastic lamellae. We conclude that the internal carotid artery is anatomically closed at a level just proximal to the carotid rete and hence has no direct involvement with cerebral blood supply in the Monodontidae. Our results confirm other investigators' work on smaller cetacean species.

In this paper we provide evidence that ectoplasmic specializations are a form of intercellular adhesion junction. Ectoplasmic specializations, found at basal junctions between adjacent Sertoli cells and at sites of adhesion between Sertoli cells and germ cells, consist of actin filament bundles sandwiched between the plasma membrane and a cistern of endoplasmic reticulum. The actin filaments in each bundle are unipolar and are hexagonally packed. The bundles are coupled to the adjacent membranes and to each other. Because ectoplasmic specializations are associated with junctional sites, they may play a role in intercellular adhesion. In this study, we report a procedure for obtaining samples enriched for ectoplasmic specializations and identify polypeptides that may be associated with ectoplasmic specializations. On SDS-polyacrylamide gels, an 83K (K = 10(3) Mr) polypeptide is specific to the ectoplasmic specialization-enriched sample, suggesting that it may be a component of ectoplasmic specializations. Other polypeptides at 38, 53, 56 and 69K also may be associated with ectoplasmic specializations. Immunoblots further indicate that fimbrin and vinculin are present in the ectoplasmic specialization-enriched fraction. In addition, immunofluorescence indicates that vinculin is associated with spermatid-Sertoli cell and Sertoli-Sertoli cell junctions. We suspect that fimbrin, an actin-bundling protein, may be involved in cross-linking the hexagonally packed actin filaments in ectoplasmic specializations while vinculin may be associated with actin-membrane linkages. If so, ectoplasmic specializations may be a new class of actin-associated junctional site. Moreover, the presence of vinculin in testicular fractions enriched for ectoplasmic specializations and at junctional sites supports the view that these structures may play a role in intercellular adhesion, possibly by stabilizing an adhesive membrane domain.

Rat Leydig cells were permeabilized and the cytoplasm partially extracted to visualize, describe, and characterize filamentous elements of the cytoskeleton. It was demonstrated by immunofluorescence microscopy that vimentin is abundant within Leydig cells. Ultrastructurally, intermediate filaments in Leydig cells were concentrated at perinuclear sites and comprised bundles that coursed through the cytoplasm. Actin was identified in Leydig cells with the F actin probe, NBD-phallacidin. Fluorescence was strongest at the cortex of the cell. With myosin S-1 subfragments, sparse actin was found positioned almost exclusively in cortical regions of the cell associated with coated pits and in Leydig cell processes.

In this study, the innervation of cerebrally related retial arteries in the narwhal Monodon monoceros was examined. Vessels were processed for the demonstration of adrenergic nerve endings by fluorescence histochemistry, and the results were confirmed by electron microscopy. Innervation of cerebrally related retial arteries was compared to that of a system situated in the haemal canal and supplying the tail. The retial arteries were poorly innervated. Adrenergic nerve endings, as indicated by fluorescence, occurred only in caudal portions of the spinal rete. Ultrastructurally, nerves were found in most retial vessels examined. However, except for arteries from caudal portions of the spinal rete, nerve numbers were few and because they occurred in outer layers of the adventitia were probably not functionally significant. In contrast, vessels in the haemal canal were well innervated. Nerve endings possessing neurotransmitter vesicles were adjacent to the smooth muscle cells. The cetacean rete mirabile, a system which supplies blood to the entire central nervous system, is apparently not under extensive nervous control, even though most reports suggest there is a relationship, possibly based on the presence of adjacent nerve trunks. Any vasomotor activity that does occur, possibly does so in response to catecholamines or other vasoactive agents circulating in the blood., (Copyright © 1981 Wiley-Liss, Inc.)

Actin filaments are concentrated in specific regions of spermatogenic cells and Sertoli cells. In spermatogenic cells they occur in intercellular bridges and in the subacrosomal space. In Sertoli cells they are abundant in ectoplasmic specializations and in regions adjacent to tubulobulbar processes of spermatogenic cells. At all of these sites, the filaments are morphologically related to the plasma membrane and+or intercellular membranes, and, as in many other cell types, are arranged in either bundles or networks. In at least two of the locations just indicated (ectoplasmic specializations and intercellular bridges), elements of the ER are closely related to the actin filaments. In tubulobulbar complexes, ER is present but is more distantly related to the filaments. Elements of the ER, when present, may serve a regulatory function. The filaments in ectoplasmic specializations and in regions adjacent to tubulobulbar processes of spermatogenic cells are suspected to be involved with the mechanism by which intercellular junctions are established, maintained, and degraded. In intercellular bridges, actin filaments may serve to reinforce and perhaps regulate the size of the cytoplasmic connections between differentiating germ cells. Filaments in the subacrosomal space may serve as a linking network between the acrosome and nucleus and may also be involved in the capping process. Because of the possibility that the actin filaments discussed before may be related to specific membrane domains involved with intercellular or interorganelle attachment, and that changes in these membrane domains are prerequisite to processes such as sperm release, turnover of the blood-testis barrier, formation of the acrosome, and coordination of spermatogenic cell differentiation, an understanding of exactly how these actin filaments are related to elements in the membrane and how this interaction is controlled is fundamental to our understanding, and perhaps our manipulating, of male fertility. I suspect that working out the molecular organization of these actin filament-containing sites and determining how their organization is controlled will be the major focus of research in this field over the next few years.

In this study, we describe the distribution of actin filaments in and around spermatids that are mechanically dissociated, in the presence and absence of exogenous trypsin, from the seminiferous epithelium of the rat. NBD-phallacidin and subfragment 1 of the myosin molecule (S-1) are used as probes for filamentous actin at the light and electron microscopic levels, respectively. The fluorescence associated with spermatids mechanically dissociated in the absence of trypsin is due to actin both in the spermatogenic cells themselves and in attached Sertoli cell ectoplasmic specializations. Fluorescence generated by labelled actin in ectoplasmic specializations occurs in linear tracts that follow the outer contour of spermatid heads. The residual fluorescence seen when trypsin is used to detach Sertoli cell fragments is diffuse and due to f-actin in the subacrosomal space. Electron microscopic data agree with the fluorescence results. This study conclusively demonstrates that Sertoli cell ectoplasmic specializations remain attached to spermatids mechanically dissociated from the seminiferous epithelium. This observation may be useful when attempting to isolate ectoplasmic specializations for biochemical analyses.

We have investigated the possibility that the complex patterns of fluorescence associated with spermatids of the ground squirrel labeled with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin (NBD-phallacidin) are due to the presence of filamentous actin within the spermatids themselves rather than to actin in attached Sertoli cell ectoplasmic specializations, as previously reported (J. Cell Biol., 100:814-825). Enzymatic treatments (trypsin, DNAase 1) freed Sertoli cell ectoplasmic specializations from spermatids and resulted in a loss, from the spermatids, of the complex fluorescence patterns, suggesting that the latter were generated by labeled actin in ectoplasmic specializations. Moreover, ectoplasmic specializations that were detached enzymatically from spermatids demonstrated the same fluorescence patterns as those emitted from spermatids in the intact or mechanically fragmented seminiferous epithelium. Most spermatids, however, do display a weak and diffuse pattern of fluorescence that changes during spermatogenesis and that is localized between the acrosomal cap and nucleus. S-1 decoration confirmed this subacrosomal localization and further demonstrated that the actin in adjacent Sertoli cell ectoplasmic specializations is arranged in a unipolar fashion. We conclude that the complex patterns of actin fluorescence associated with mechanically isolated spermatids are a superimposition of both Sertoli cell and germ cell actin; however, the latter is either poorly detected or not detected at all when Sertoli cell ectoplasmic specializations overlie the germ cells.

The effects of ethanol consumption on the morphology of the seminiferous epithelium, with particular emphasis on Sertoli cell ultrastructure, were examined during and following pubertal development. Sprague-Dawley rats were maintained on chronically high levels of ethanol for 7 weeks beginning at 29 days of age. Animals in Group 1 were fed a liquid diet containing ethanol (36% ethanol-derived calories) ad libitum. Group 2 animals were paired with animals in Group 1 and fed a liquid control diet in the amount consumed by their ethanol partners (g/kg body wt/day). Animals in Group 3 were fed Purina rodent chow ad libitum. Blood samples were collected at 60 days for determination of plasma testosterone levels. On day 79, each epididymis, the adrenals and the right testis were removed from anesthetized animals and weighed; the left testis was removed and processed for light and electron microscopy. Blood alcohol levels were consistently high throughout the feeding period, averaging 272.6 +/- 9.7 mg/100 ml at 1900 hours (1 hour after lights off) and 178.8 +/- 20.8 mg/100 ml at 1330 hours. Testosterone levels were lower in ethanol-consuming animals than in pair-fed or control subjects. Testis weight was also somewhat reduced in ethanol-consuming animals; however, when adjusted for body weight, relative testis weights were found to be increased in ethanol and pair-fed animals. Epididymal weights were reduced in both ethanol and pair-fed animals. Relative adrenal weights were increased by ethanol. The most dramatic effect of ethanol consumption was on the morphology of the seminiferous tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

Filaments about 6-7 nm in diameter were seen associated with germ cell intercellular bridges in detergent-permeabilized cells treated with tannic acid. Approximately 40-50 filaments were present subjacent to the bridge density. Filaments encircled the bridge channel in a manner similar to contractile ring actin filaments of dividing cells. NBD-phallacidin and myosin S-1 subfragments were employed to demonstrate that the filaments observed at intercellular bridges are actin. Intratesticular injection of a single dose of cytochalasin D, a specific inhibitor of actin filaments, caused certain intercellular bridges of spermatids to open within 3 hr after injection, leading to the production of symplasts. During bridge opening, remnants of bridge densities were gradually incorporated into the lateral aspect of the plasma membrane of the symplast. Thus actin, present in bridge structures, appeared to participate in maintaining certain intercellular bridges. A model of intercellular bridge structure is presented.