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The Sertoli Cell Occluding Junctions and Gap Junctions Developing Mammalian Testis’ NORTON Department

B. GILULA,~

DON W. FAWCETT,

of Anatomy, and Laboratory of Human Harvard Medical School, Boston, Accepted

December

AND AGUSTIN

Reproduction Massachusetts

in Mature and

AOKI~

and Reproductive 021.l5

Biology,

11,1975

Special occluding junctions between Sertoli cells near the base of the seminiferous epithelium are the structural basis of the blood-testis permeability barrier. In micrographs of thin sections, multiple punctate pentalaminar contacts between apposed membranes are observed in the junctional regions. In freeze-fractured mature testis, the junctional membranes exhibit up to 40 parallel circumferentially oriented rows of intramembrane particles preferentially associated with the B-fracture face, but with complementary shallow grooves on the A-face. Short rows of particles may remain with the A-face resulting in discontinuities in the B-face particle rows. In addition, elongate aggregations of particles of uniform size (-70 A) arranged in one or more closely packed rows are occasionally found adjacent to the linear depresssions on the A-face of the Sertoli junction. These are interpreted as atypical gap junctions. In immature testis, occluding junctions are absent but typical gap junctions are common. These gradually disappear. In the second postnatal week, linear arrays of particles appear on the B-face. Initially meandering and highly variable in direction, these gradually adopt a consistent orientation parallel to the cell base. The establishment of the blood-testis barrier appears to be correlated with this reorganization of the intramembrane particle rows. Sertoli junctions were shown to be resistant to hypertonic solutions that rapidly dissociate junctions of other epithelia. Sertoli junctions thus differ from other occluding junctions in their (1) basal location, (21 large number of parallel particle rows, (3) absence of anastomosis between rows, (4) preferential association of the particles with the B-face, (5) intercalation of atypical gap junctions, (61 unusual resistance to dissociation by hypertonic solutions. INTRODUCTION

(Farquhar and Palade, 1963). In the past several years, the development of the Electron microscopic studies of thin secmethod of freeze-fracturing has permitted tions from a great variety of mammalian tissues have led to the recognition of three examination of the differentiations within basic types of membrane specialization on the plane of the membrane at these juncthe contiguous surfaces of epithelial cells. tional specializations and has revealed There are those providing for occlusion of considerable variation within the three intercellular clefts and regulation of epi- types (Friend and Gilula, 1972; McNutt thelial permeability (zonula occludens); and Weinstein, 1973; Gilula, 1974; Staethose involved in cell-to-cell communica- helin, 1974). It has been possible in some tion (nexus or gap junction); and those instances to relate these structural variaconcerned with maintenance of tissue tions to special properties of the junctions cohesion (desmosome or macula adherens) and to tissue-specific functions (Gilula, Beeves, Steinbach, 1972; Claude and Good’ Supported by Research Contract NOl-HD-9-2107 enough, 1973). To date there have been few from the Center for Population Research, National studies correlating these membrane speInstitute of Child Health and Human Development. cializations with the function of epithelia 2 Dr. Gilula’s present address is The Rockefeller in reproductive organs. University, New York, New York 10021. :I Supported on a grant from the Ford Foundation. The mammalian testis produces testicu142 Copyright All rights

Q 1976 by Academic Press, Inc. of reproduction in any form reserved.

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puberty concurrently with (1) the establar fluid and spermatozoa as its exocrine lishment of the permeability barrier, (2) products, but it is also an endocrine gland the beginning of fluid secretion, and (3) secreting androgenic steroid hormones, the development of a lumen in the seminifwhich serve locally to maintain spermatogenesis and which act at a distance to erous tubules. It was suggested, therefore, that these concurrent events are probably maintain the accessory reproductive all dependent upon the presence of occludglands and secondary sexual characterising junctions near the base of the epithetics. The endocrine function of the testis and Dym, 1973). resides mainly in the interstitial cells of lium (Vitale, Fawcett, In the present study, we have combined Leydig while the seminiferous tubules are information provided by thin sections with the site of production of the spermatozoa. The tubules are bounded by a thin layer of that available in replicas of freeze-fractured mature testis in order to define more epithelioid contractile cells (myoid cells) precisely the characteristics of the juncand are lined by the seminiferous epithetional specializations of Sertoli cells and to lium. This exceptional stratified epithecompare them with junctions in other lium is composed of two distinct categories of cells: a fixed population of columnar types of epithelia. Experiments were carSertoli cells which extend from the base to ried out to determine whether the Sertoli the lumen, and intercalated between these junctions can be dissociated by exposure to supporting cells is the mobile population of hypertonic solutions which are known to germ cells which proliferate near the base open the blood-brain barrier. In addition, and gradually move upward in the epithethe seminiferous tubules of young animals lium as they differentiate into spermatohave been examined prior to establishzoa. The spermatozoa are then released ment of the blood-testis barrier in order to into the lumen. gain some insight into the development of The necessity for upward mobility of the the Sertoli junctional specializations, and developing germ cells with respect to the into the reorganization of the membranes stationary population of supporting cells that is associated with the onset of sperdoes not permit the formation of enduring matogenesis. Atypical communicating or specializations for cell-to-cell attachment gap junctions are also present between the such as are commonly found in other epi- bases of the Sertoli cells and the changes thelia. Therefore in electron micrographs in this type of junction during developof seminiferous epithelium, no juxtalument of the blood-testis barrier have been minal occluding junctions are found, and examined. Some preliminary observations neither desmosomes nor typical gap juncfrom this study have been published elsetions are observed on the interface be- where (Fawcett, 1974a). tween germ cells and Sertoli cells (FawMATERIALS AND METHODS cett, 1974a, 197413). There are, however, unique occluding junctions between adjaMature Sprague-Dawley male rats were cent Sertoli cells near the base of the epiobtained from Charles River Breeding thelium (Fig. 1). That these are the morLaboratories, North Wilmington, Mass., phological basis of the blood-testis permeafor these studies. For studies on the develbility barrier in the mature testis has been opment of the blood-testis barrier in imdemonstrated in several studies using mature tissue, samples were taken from electron-dense probes of the extracellular male Sprague-Dawley rats (Charles River space and electron microscopy of thin sec- Breeding Laboratories) at 10, 11,14,15, 17, tions (Fawcett et al., 1970; Dym, 1973; 18, and 20 days after birth. Also included Aoki and Fawcett, 1975). These junctions are observations on several mature guinea have been shown to appear in the rat at pigs.

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FIG. 1. Diagram illustrating the location of the occluding Sertoli junctions and presenting their structural components. The large arrow indicates the path of membrane cleavage in the freeze-fractured illustrations presented in subsequent figures of this paper. [From Fawcett (1975). “Handbook of Physiology.” Vol. 5: Endocrinology, Chap. 2. Amer. Sot. Physiol.]

Tissues for thin sections were fixed by local injection of 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) into the testicular tissue immediately after death. After lo-15 min, the testes were removed from the animal and the tissue was minced into small pieces. The tissue pieces were then fixed for 2-4 hr at room temperature in 5% glutaraldehyde buffered with 0.1 M sodium cacodylate (pH 7.3). This fixation was followed by treatment with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 hr at room temperature, treatment with uranyl acetate in Verona1 acetate buffer for 1 hr at room temperature, dehydration with ethanol, and Epon 812 embedding.

Tissue for freeze-fracturing was either unfixed prior to freezing, or it was treated with 5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 30-40 min at room temperature. The prefixed tissue was subsequently treated with 25% glycerol in 0.1 M cacodylate buffer (pH 7.3) for 2-24 hr at 4°C prior to freezing. Both unfixed (unglycerinated) and prefixed (glycerinated) tissues were rapidly frozen in Freon 22 and stored in liquid nitrogen prior to freezefracturing. Samples were freeze-fractured at -115°C in a Balzers BM360 apparatus. Carbon-platinum replicas were obtained and cleaned in bleach and water prior to mounting on uncoated copper grids. All electron microscopic observations

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were made with a Siemens Elmiskop 1A and a Philips 300. All freeze-fracture images have been mounted so that the shadow direction is from the bottom to the top of the micrograph. In this manuscript the fracture faces are designated as outer membrane halves (Fracture Face B) and inner membrane halves (Fracture Face A). The A face corresponds to the P face, and the B face corresponds to the E face in the nomenclature system that has been recently proposed (Branton et al. (1975), Science 190, 54-56). Experimental procedures. In experiments intended to dissociate the Sertoli junctions, 15 adult and 8 immature 13-dayold male rats were studied. Testes of adults were exposed through a scrotal incision and the internal spermatic artery was cannulated with a 25-gauge needle connected to a venoclysis set. Through this system, hypertonic solutions were perfused in various experiments in the following concentrations: lithium chloride 0.25 M, 0.5 M, and 0.76 M; urea 2 M; and sucrose 0.5 M. The perfusions with these solutions were continued for 1 to 10 min and followed immediately by the fixative, consisting of 4% glutaraldehyde in 0.1 M scollidine buffer containing 20 mM calcium chloride. When lithium chloride or urea were perfused for more than 2 or 3 min, interstitial edema developed to such an extent that it was necessary to incise the tunica albuginea and relieve the pressure in order to insure penetration of the perfused fixative. Perfusion through the testicular artery in immature rats is not possible and perfusion of hyperosmotic solutions through the aorta does not consistently result in irrigation of the testis. It was necessary therefore to simply transect the testis creating three or four pieces and to incubate these in 0.75 M lithium chloride for 1 to 5 min. The tissues were then transferred to the fixative and processed for light and electron microscopy as described above.

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To determine whether development of the Sertoli junctions depended upon the presence of germ cells, a rat was given a diester of methane sulphonic acid (Busulphan) during pregnancy to selectively destroy the stem cells of her male offspring (Jackson, 1965). These animals were used as young adults for experiments involving exposure of seminiferous tubules to hypertonic solutions. RESULTS

In electron micrographs of mature testis, the junctional complexes between adjacent Sertoli cells are located either just above the basal lamina or between lateral processes of these supporting cells that arch over the spermatogonia (Figs. 1 and 2). Although these junctions have previously been described in some detail by several investigators (Brokelmann, 1963; Flickinger and Fawcett, 1967; Nicander, 1967), a review of their salient features is a necessary background for the description that follows of the specializations within the plane of their membranes. In thin sections they are characterized by (1) the presence of an extensive series of focal, pentalaminar contacts where the opposing membranes appear to fuse (Figs. 12 and 13); (2) occasional small septilaminar areas that resemble gap junctions but are more variable in linear extent (Fig. 8). (3) Discrete bundles of filaments, usually found in the superficial cytoplasm of both cells subjacent to the series of focal membrane contacts (Figs. 2 and 12). These course parallel to the cell surface and to the basal lamina. (4) Deep to the layer of filaments in both cells are cisternae of the endoplasmic reticulum oriented parallel to the cell boundary. These latter are irregularly fenestrated and therefore present discontinuous profiles of varying length (Figs. 2 and 4). They often bear ribosomes on the membrane toward the cell body but are agranular on the side adjacent to the filament bundles. In tubules exposed to lanthanum, perox-

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idase, or other probes of the extracellular space, these basal junctional complexes between Sertoli cells constitute an effective barrier to deeper penetration of the epithelium (Dym and Fawcett, 1970; Aoki and Fawcett, 1975). These unusual specializations near the base of the seminiferous epithelium are therefore a kind of occluding junction functionally analogous to the zonulae occludentes or tight junctions found near the luminal surface of many other epithelia (Farquhar and Palade, 1963). There are, however, significant differences when the two types of junction are examined by freeze-fracturing. In replicas of planes of cleavage near the base of the seminiferous epithelium, the Sertoli junctions are identifiable as multiple rows of intramembrane particles coursing circumferentially around the cell parallel to the basal lamina (Figs. 6 and 7). The rows usually are not interconnected but are roughly parallel and show considerable variation in their spacing (40 to 300 nm). The rows are not comprised of homogeneous, closely packed particles, nor are the particles fused into continuous ridges (as they are in a typical zonula occludens). Instead, they are made up of particles that vary in size from 65 to 110 A. They are preferentially associated with the B-face of the membrane with the unspecialized regions between rows being smooth and virtually devoid of particles. The A-fracture face exhibits an extensive series of shallow, continuous grooves that complement the rows of particles on the B-face. The

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preferential association of the particles with the B-face is not absolute for there are frequent discontinuities of varying length in the rows on this face, and the linear depressions on the A-face frequently contain short rows of particles that correspond to the hiatuses in the rows on the Bface (Figs. 6 and 7). The particles found at irregular intervals in the grooves on the Aface seem more homogeneous in size (90100 A) than those of the B-face and often fuse to form short ridges (Fig. 15). In some regions, this particulate component may occupy from 25 to 50% of the linear dimensions of the grooves on the A-face. Thus, although the rows of particles appear interrupted on both fracture faces, they are probably continuous in the intact membrane. The unspecialized regions of the Aface show the usual heterogeneous population of random particles (Figs. 10 and 15). It is important to note that the fracture face disposition of the particulate components of the Sertoli junctions is not detectably different in specimens which are frozen without prior fixation. These extensive junctions are sufficiently novel and distinctive in their structure and properties to deserve designation by a term other than zonula occludens. Because they appear to be unique to the supporting cells of the testis, the term “Sertoli junction” seems appropriate. A feature of Sertoli junctions in thin section which does not seem to have been reported heretofore is the presence of focal contacts and apparent fusion between the -

FIG. 2. Electron micrograph of a portion of the boundary of two Sertoli cells showing a typical Sertoli junction in the mature testis. Cisternae of the endoplasmic reticulum parallel to the cell membranes have ribosomes on the side toward the cell body but none on the side toward the plasmalemma. x 51,750. FIG. 3. Boundary of two Sertoli cell precursors from a lo-day postnatal rat testis. Occluding Sertoli junctions have not yet formed but gap junctions (GJ) are common. x 63,000. FIG. 4. Micrograph of a developing Sertoli junction from a 20-day postnatal rat testis. Cisternae of the endoplasmic reticulum have taken up a position in each cell parallel to the surface membrane. x 36,000. FIG. 5. Sertoli junction from a mature testis. Cisternae of the reticulum associated with the junction present long profiles. The plane of section is parallel to the bundles of filaments. In this plane the intercellular cleft may be narrowed but sites of membrane fusion are not observed. When sectioned transverse to the filament bundles, multiple sites of membrane fusion can be seen (see Figs. 12 and 13). x 61,200.

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membranes on opposite sides of the subsurface cisternae (Figs. 8 and 12). These bear a superficial resemblance to the sites of membrane fusion across the intercellular cleft, but they have not been identified in freeze-fracture preparations, and it is not known whether they are punctate or linear. It is possible that they represent an early stage in the formation of a fenestration in the cistern but they have not been noticed in cisternae of the endoplasmic reticulum in other cell types or in other locations in the Sertoli cell. Another reason for suspecting that they may have special significance in the physiology of the junctional complex is the observation that they do not appear to be random in their distribution but seem always to occur adjacent to one of the bundles of junctional filaments and not opposite the intervals between successive filament bundles. This relationship deserves further study. Interpretations differ as to the relation of the anastomosing ridges of occluding junctions to the lipid bilayers of the opposing membranes. Some investigators envision a single network of rodlike elements (fibrils) bridging the intercellular space and projecting into the hydrophobic interior of both membranes (Wade and Karnovsky, 1974). According to others, the pattern of ridges or rods is duplicated in both membranes and the two are in register (Chalcroft and Bullivant, 1970). Membrane fusion is attributed to firm adherence of the matching ridges in opposing membranes (Staehelin, 1974). In favorable thin sections of Sertoli junctions, pairs of globular pale areas, one in each membrane, can be occasionally resolved at sites of membrane fusion (Fig. 13). This appearance suggests that the linear particle ar-

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rays seen in freeze-fracture preparations of Sertoli junctions are duplicated in the two interacting membranes, and that the pairs of clear areas observed in thin sections represent negative images of the apposed particle rows. Why they appear as areas of lower rather than greater density is not readily explained. Another type of membrane specialization, the gap junction, is observed on the A fracture face intercalated among the particle rows of the Sertoli junctions. It is neither common nor conspicuous but may be of considerable functional importance. It is usually found in close association with the linear depressions on the A-face of the Sertoli junctions (Fig. 15) and consists of particles of uniform size (-70 A) aggregated in rectilinear arrays one to five rows in width. The rows are of variable but rather limited length, and the particles are closely packed with a center-to-center spacing of about 100 A (Figs. 9 and 15). The rows are often solitary (Fig. 10) but, in many instances, there are several in close proximity. Complementary B-face pits have not been observed in these regions. On the basis of the uniform size of the particles, their spacing, their association with the A-face, and the observation of septilaminar segments in thin sections (Fig. 8), these rows of particles are interpreted as an unusual form of gap junction. This identification is strengthened by the observation of numerous typical gap junctions between Sertoli cells in immature testes (Fig. 3). Thus, although junctional specializations are essentially absent on the interface between supporting cells and the differentiating germ cells in the adluminal two-thirds of the epithelium, the Sertoli

FIG. 6. Replica of a freeze-fractured Sertoli junctional membrane showing multiple rows of intramembrane particles. The linear arrays of particles course circumferentially around the base of the cell and are roughly parallel. Unlike the juxtaluminal occluding junctions of other epithelia, the rows do not anastomose and they are preferentially associated with the B-face (i.e., the outer half membrane). Where the rows on the B-face appear discontinuous, the missing segments are associated with the complementary A-face element, as can be seen at the lower right. x 78,500.

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cells near the base of the epithelium have occluding junctional complexes of a unique type that constitute the morphological basis of the blood-testis barrier (Dym and Fawcett, 1970; Fawcett, 1974b). They also have atypical gap junctions which may provide for communication and the coordination of events along the length of the tubules.

for short distances. These areas of close contact appear to be typical septilaminar contacts or gap junctions (Fig. 3). Profiles of endoplasmic reticulum are sparse and show little tendency to be oriented parallel to the plasmalemma. Rarely in short segments where the intercellular space is narrowed one observes subplasmalemmal dense material superficially resembling that seen at desmosomes or fasciae adherSertoli Junctions in Immature Testis entes. By 15 days, the areas of narrowing In the early postnatal period, the sup- of the interspace between supporting cells porting cells of the seminiferous cords in are more extensive and often have short the rat divide actively. Proliferation slows cisternal profiles of endoplasmic reticulum rapidly after the first week and no mitosis coursing parallel to the surface on one or or incorporation of tritiated thymidine oc- both sides of the cell boundaries. Focal curs after the 15th day (Clermont and pentalaminar junctions are only rarely Percy, 1957; Steinberger and Steinberger, seen in the relatively extensive areas of 1971). The Sertoli cells are therefore very narrowing of the intercellular cleft. At 20 long lived, persisting throughout the life of days, desmosomelike plaques persist in the animal. In the first 2 weeks of life, small numbers but now Sertoli cell juncthere is no permeability barrier. Lantions are common in the basal region of the thanum and other electron opaque extraepithelium, and they exhibit all of the cellular tracers penetrate throughout the characteristic features of these junctions seminiferous cords, and occluding Sertoli in mature testis except that the focal sites junctions are not seen in electron microof membrane fusion are fewer and the cisgraphs of thin sections (Vitale et al., 1973). ternal profiles are shorter and often more Between the 15th and 20th days, Sertoli dilated. junctions appear and a blood-testis permeIn replicas of freeze-fracture preparaability barrier is established. Then with tions of 15-day rat testis, the intramemthe onset of fluid secretion, the seminiferbrane rows of particles are already present ous cords are transformed into tubules. It and are very extensive (Fig. 14). Their was thought to be of interest to study the fracture-face affinities are similar to those seminiferous epithelium by freeze-fracturof the junctions in mature testis but there ing during this significant transitional pe- are two striking organizational differriod. ences. The particle rows are not aligned in In thin sections of lo-day rat testis, one parallel array to form a broad zonula pardoes not encounter typical Sertoli juncallel to the cell base. Instead, the rows are tions. The membranes of the supporting notably sinuous and meandering in their cells for the most part appear unspecialcourse. Some end abruptly, others are inized and separated by an interspace of terconnected with neighboring rows to about 200 A but occasionally the memtrace a highly irregular plexiform pattern branes come into much closer association on the B-face. Even though the rows may FIG. 7. Freeze-fracture image of a Sertoli junction, again illustrating the large number of linear arrays of particles on the B-face of the Sertoli cell membrane. A few individual particles or short rows are also seen on corresponding ridges of the A-face. Note the striking complementarity between the A-face and B-face elements. It is not uncommon for these broad occluding junctions to have 40 to 50 rows of intramembrane particles. x 72,000.

FIG. 8. Micrograph of a Sertoli junction region in thin section showing a typical septilaminar gap junctional profile intercalated between successive sites of membrane fusion. The gap junctions are infrequently observed in thin sections of mature testis. x 160,000. FIG. 9. A small area of the A-face of a Sertoli junction showing rows of particles of uniform size (white arrows) coursing parallel to discontinuous rows of larger particles or ridges (bIack arrows). The former represent gap junctions, while the latter are segments of particle rows of the Sertoli junction which have adhered to the A-face. x 150,000. FIG. 10. A small area of the A-face showing, above, a gap junction consisting of a single row of particles and, below, several similar linear gap junctions. A delicate line can be discerned adjacent to each of these that represents the shallow groove in the A-face complementary to the B-face particle rows of the Sertoli junction. The linear gap junctional particles are characteristically associated with the Sertoli junction element, and typical gap junctional plaques have rarely been observed in the mature testis. x 183,000. 152

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have a prevailing orientation, their direction may be vertical or oblique with respect to the axis of the cell. This is in contrast to the consistent horizontal, circumferential orientation of the parallel rows in the Sertoli cell junctions of the mature testis. Our investigations indicate that in the immature testis, differentiation of intramembrane rows of particles in the Sertoli cell membranes is well advanced before an effective permeability barrier can be demonstrated by extracellular tracers. The paucity of focal pentalaminar contacts in thin sections at stages when the particle rows are rather extensive in freeze-fractured preparations suggests the possibility that, at early stages of junction development, the patterns traced by the particle rows of apposed membranes may not yet be in register, and multiple circumferential lines of membrane fusion may only develop later when the particle rows become rearranged in a broad circumferential band parallel to the base of the epithelium. Establishment of an effective barrier therefore does not coincide with the first appearance of rows of particles in the membrane, but awaits their realignment in register and their orientation athwart the path of substances penetrating the epithelium from its base. In the immature testis, conspicuous oval or elongate gap junctions of varying size are frequently observed on the boundaries of the Sertoli cell precursors. These particle aggregates exhibit the typical hexagonal close packing but other patterns are seen occasionally. As development progresses, these typical gap junctions become less numerous and ultimately disappear entirely in the adluminal portion of the epithelium. After the development of the basal permeability barrier, the gap junctions remaining are of small size, atypical linear configuration, and they are confined to the areas between the particle rows of the Sertoli junctions. These A-face particle aggregates may rarely form small

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isodiametric plaques but more often they consist of one to four rows of closely packed particles disposed along the A-face grooves that are complementary to the B-face particle rows of the Sertoli junctions. These linear or rectilinear gap junctions appear to be more abundant during the development of the Sertoli junctions than they are in mature testis (Fig. 14). Stability tonic

of the Sertoli Solutions

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The communicating junctions (nexuses) between smooth and cardiac muscle cells have been reported to come apart after perfusion with hypertonic sucrose (Barr et al., 1965; Dreifuss et al., 1966). Similar treatment results in dissociation of the hepatic gap junctions in a matter of seconds, and the zonulae occludentes at the commissures of the bile canaliculi are also separated (Goodenough and Gilula, 1974). The blood-brain permeability barrier is rapidly opened by perfusion with hypertonic urea or lactamide (Rapoport and Thompson, 1973). We were interested therefore to determine whether the junctions maintaining the blood-testis barrier were equally susceptible to dissociation by hypertonic solutions. In earlier publications, attention has been drawn to the fact that the Sertoli junctions are commonly located on the boundary between supporting cell processesthat overarch the spermatogonia. As a consequence of this location, they create two distinct compartments within the epithelium: a protected adluminal compartment containing the spermatocytes and spermatids, and a basal compartment containing the spermatogonia. The stem cells, therefore, are outside of the blood-testis barrier and thus are directly exposed to conditions prevailing in the interstitium of the testis (Dym and Fawcett, 1970). In experiments intended to dissociate the Sertoli junctions, rats were perfused intravascularly with 0.25 to 0.76 M lithium chloride, and this was followed after 1 to 3 min

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(Fig. 17). The Sertoli junctions are eviby perfusion of the glutaraldehyde fixadently unusually resistant to osmotic distive. Histological sections of mature rat ruption and for some time after perfusion, testis after this treatment present a very the blood-testis barrier affords protection interesting appearance (Fig. 16). The hyof the cells in the adluminal compartment pertonic solution rapidly escaping from the against a steep osmotic gradient. interstitial blood vessels results in a After a few minutes of perfusion with marked shrinkage of the Leydig cells and hypertonic solutions, the resulting interboundary tissue. The seminiferous tubules stitial edema makes it impossible to obtain retain their normal circular cross-secadequate fixation by further perfusion tional profile. The basal portion of the Serwith glutaraldehyde. Study of later effects toli cells is darkly stained, due to local condensation of their cytoplasm by os- was therefore more difficult. It is obvious, however, that at longer time intervals, motic withdrawal of water (Fig. 17), but of cells does ultimately extend these cells remain firmly adherent to shrinkage throughout the epithelium. The results of the basal lamina. The spermatogonia, however, are almost invariably detached these experiments, nevertheless, show that the Sertoli junctions are exceptionally from the basal lamina and remarkably resistant to osmotic disruption, and since shrunken, so that the spaces they northey are impermeable to lithium salts, the mally occupy appear quite empty (Figs. 16 and 17). germ cells in the adluminal compartment The cellular contents of the basal comare protected by a continuous barrier of partment of the epithelium are thus supporting-cell processes and resistant highly vulnerable to osmotic damage and junctions that separate them from the are rapidly shrunken and condensed, probasal compartment of the tubule and from viding a dramatic demonstration of the the interstitium of the testis. size and configuration of this compartElectron micrographs of such preparament. In contrast, the spermatocytes and tions show that the lymphatic endothespermatids occupying the adluminal comlium and myoid cells comprising the partment are not affected, at short time boundary tissue of the tubules still form intervals, by the hypertonic conditions coherent sheets of cells, but they are very prevailing in the interstitium of the testis thin and the cytoplasm extremely dense FIG. 11. At high magnification, the freeze-fracture elements of the Sertoli cell junction in the mature testis are characterized by the B-face linear array of particles and the A-face complementary grooves (arrows). In some regions, ridgelike particles of the B-face components occupy the complementary grooves. A variety of nonjunctional particles are present between the Sertoli junction specializations; these are most prominent on the A-face. x 150,000. FIG. 12. Thin-section micrograph of Sertoli junctional region in mature testis that demonstrates the characteristic elements of the junctional region: multiple sites of membrane fusion (at the smaller arrows) and clusters of microfilaments. In addition, punctate fusions between membranes of the associated cisternae of endoplasmic reticulum are also apparent in this image (at larger arrows). x 100,000 (micrograph by M. Dym). FIG. 13. High magnification of the sites of Sertoli junction membrane fusion. Round pale areas interrupt the typical trilaminar image of both membranes at the sites of fusion (see arrows). These profiles may represent cross-sectional images of the particle rows that are seen within the plane of the membranes in freeze-fracture preparations x 200,000 (micrograph by M. Dym). The inset presents alternative interpretations of the membranes and their internal specializations at occluding junctions of other epithelia (modified from J. B. Wade and M. J. Karnovsky, J. Cell. Biol. 60,168-191). The image presented here does not suggest rows of particles shared by the two membranes (B) but favors interpretation (A) in which corresponding intramembranous specializations in the opposing membranes are in register and adhere obliterating the intercellular cleft. This is in agreement with a model proposed by Chalcroft and Bullivant (19701. This finding for the Sertoli junction does not exclude a different interpretation for other occluding junctions.

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(Fig. 18). The cytoplasm at the base of the Sertoli cells is also much darker than usual, and the organelles and inclusions are crowded together. The nucleoplasm which is normally homogeneous and essentially devoid of heterochromatin is dark and mottled by small clumps of chromatin after exposure to hypertonic lithium chloride. The empty-appearing chambers of the basal compartment observed in histological sections are found, in electron micrographs, to be bounded on the abluminal side by an intact basal lamina supported by a sparse stroma of collagen fibrils and the underlying myoid cells. On the adluminal side, the enlarged spaces of the basal compartment are bounded by thin condensed cytoplasmic processes of the neighboring Sertoli cells (Fig. 18). Intact Sertoli junctions can often be seen joining these processes (Fig. 19). Shrunken spermatogonia are occasionally encountered in the cavities of the basal compartment. They appear to have come away from the basal lamina with an intact membrane. Their cytoplasm is highly condensed and the nucleus contains conspicuous irregular masses of condensed chromatin. The basal compartment is otherwise empty except for a few myelin forms of hydrated phospholipid. The spermatocytes and spermatids on the adluminal side of the barrier show little or no change in volume or in the density of their cytoplasm (Figs. 18 and 19). The chromosomes are slightly more condensed and more distinct in their outline than in untreated testis. Perfusion with 0.5 M sucrose which has been shown to be very effective in breaking tight junctions in the liver causes no disruption of the Sertoli junctions. Only after prolonged perfusion (5 to 10 min) are

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the changes comparable to those observed after l-3 min of perfusion with lithium chloride. Inclusion of trypan blue in the perfusate suggests that the interstitial capillary endothelium is relatively less permeable to sucrose. It might be argued that the apparent confinement of the early damage to the outer part of the tubules with the more centrally situated cells remaining normal in appearance, is simply an expression of a relatively slow rate of penetration. That this is not so was demonstrated by similar experiments on immature rats. The bloodtestis barrier is established between the 15th and 18th days after birth. When testes of rats 13 days of age (Fig. 20) were exposed to hypertonic lithium chloride, peripheral cavities corresponding to the basal compartment were not formed. Instead, cells were shrunken and intercellular clefts were expanded throughout the seminiferous cords (Fig. 21). The fact that the barrier normally develops at a time when the spermatogonia take up a basal position in the cords shortly before the onset of spermatogenesis suggested the possibility that the gonocytes or spermatogonia might play some role in induction of the Sertoli junctions. It was of interest, therefore, to examine testes of adult rats that had been exposed in utero to Busulfan. Seminiferous tubules in these animals consisted of Sertoli cells only. Nevertheless, they had developed typical Sertoli junctions. When exposed briefly to hypertonic lithium chloride solution, cavities developed between the shrunken bases of the Sertoli cells, while the adluminal portion of the epithelium showed relatively little damage. In physiological experiments measuring

FIG. 14. Freeze-fracture preparation of the junctional area of a Sertoli cell from an immature rat (15 days) before an effective permeability barrier is established. At this stage of development, the rows of intramembrane particles pursue a meandering course, frequently intersect, and are not yet consistently oriented parallel to the basal lamina. This field shows mainly fracture-face A. Especially noteworthy is the large number of gap junctional particle aggregates (at the arrows). The large round or oval plaques characteristic of gap junctions in many tissues are not present. Also, these small elongate gap junctions are rarely observed in the mature testis. x 50,000.

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the rate of penetration of various substances from blood plasma into rete testis fluid and testicular lymph (Setchell, Voglmayr, and Waites, 1969), urea was one of the substances that readily traversed the blood-testis barrier. It was of interest therefore to perfuse rat testis with hypertonic urea to see whether penetration of this substance would open the Sertoli junctions . After perfusion with urea the histological appearance of the testis was very different from that observed after hypteronic lithium chloride. There was some condensation and shrinkage of the Leydig cells, but no significant expansion of the basal compartment or damage to the spermatogonia, which remain attached to the basal lamina. The Sertoli cytoplasm in electron micrographs was not condensed but instead contained many large clear vacuoles possibly arising by distension of the elements of the endoplasmic reticulum. The Sertoli junctions appeared to be unaltered. It seems likely, therefore, that urea penetrates into the lumen of the seminiferous tubules by passing through the Sertoli cells and not by traversing the Sertoli junctions and intercellular clefts of the seminiferous epithelium. The osmotic effects of hypertonic urea appear to be mitigated by reason of its ability to enter the cells. DISCUSSION

The cells of many epithelia are linked by a circumferential tight junction or zonula occludens on their lateral boundaries just beneath the free surface. This specialization of the membranes of adjoining cells seals the intercellular clefts and largely

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prevents penetration of the epithelium by diffusion from the lumen. The permeability barrier thus formed enables the cells of absorptive epithelia to create intercellular osmotic gradients that move fluid and electrolyte from the lumen to the underlying tissue and also serves to maintain differences between the composition of tissue fluid and fluid in the lumen of epitheliallined tubules. This paper has described the unique type of occluding junction in the seminiferous epithelium which has been identified as the principal structural component of the blood-testis permeability barrier (Fawcett et al., 1970). The existence of this barrier has important consequences for testicular function. Situated near the basal lamina of the seminiferous tubules, it divides the epithelium into a basal compartment containing the spermatogonia and an adluminal compartment containing the more advanced stages of germ-cell development. It is believed that this compartmentation of the epithelium by occluding junctions serves to isolate the developing germ cells from the general extracellular space of the testis, permitting the Sertoli cells to maintain in the adluminal compartment a microenvironment favorable for the continuing differentiation of the germ cells (Dym and Fawcett, 1970; Fawcett, 1974b). It is also speculated that maintenance of a barrier at the base of the epithelium may be essential for its secretory function, making it possible to create a standing osmotic gradient in the adluminal compartment that would tend to move fluid across the epithelium into the lumen of the tubules (Setchell, 1970; Fawcett, 1975). And finally, the intraepithelial permeability

FIG. 15. In the immature rat testis (15-day postnatal), there are distinctive particle aggregates on the Aface, in addition to the linear arrays of particles on fracture-face B. These aggregates are elongate structures composed of one to five rows of closely packed particles. They tend to be located close to, and sometimes sequestered between, the grooves corresponding to the particle rows on the B-face of the Sertoli junction. From the uniform size of the particles in these aggregates, their close packing, and their association with the A-face, it is inferred that these represent the gap junctional membrane specializations in this tissue. As development continues, these specializations presumably give rise to the linear gap junctions that are present in the mature testis. x 150,000.

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barrier is probably important for the protection of the germ-cell line from bloodborne noxious agents and for impounding within the tubules antigenic products of postmeiotic germ cells that might otherwise reach the blood stream and induce an autoimmune response (Johnson, 1970). The Sertoli junction combines certain characteristics of the septate junction of invertebrates with those of the vertebrate tight junction. The membrane particle rows do not anastomose but maintain a parallel orientation much like that of the septa of septate junctions (Gilula et al., 1970). At the same time, the Sertoli junction definitely provides an occluding barrier that is the result of multiple membrane to membrane fusions, much as in the familiar tight junction (Wade and Karnovsky, 1974; Staehelin, 1973). Although the Sertoli junctions share certain physiological properties with the common zonula occludens, the freeze-fracture observations reported here have revealed several significant differences. In replicas, typical juxtaluminal zonulae occludentes appear either as a continuous bandlike meshwork of branching and anastornosing thin ridges on the A-fracture face or as a corresponding pattern of shallow grooves on the B-face (Staehelin et al., 1969). The distinguishing features of the Sertoli junction, on the other hand, are that (1) they are located near the base of the epithelium; (2) the sites of membrane interaction are not ridges but rows of closely packed intramembrane particles of varying size; (3) the rows do not anastomose extensively; (4) the aligned particles are preferentially associated with the Bfracture face, but segments of the particle rows may come away with the A-face; (5)

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the number of parallel lines of membrane fusion is far greater (up to 50) than in any other occluding junction described to date. This latter feature is probably very significant. Claude and Goodenough (1973) have recently correlated the “tightness” or “leakiness” of epithelia as determined by transepithelial resistance, with the apicobasal depth of the zonule seen in freezefracture preparations. Some of the more leaky epithelia had only one or two rows of membrane interaction, whereas the tightest junctions for which data are available were somewhat more than 0.5 pm in depth and consisted of about eight tiers of sealing strands. No measurements of transepithelial resistance are available for seminiferous epithelium but if one can extrapolate from the correlations of Claude and Goodenough, the finding of up to 50 parallel lines of particles in Sertoli junctions in a band several microns broad would suggest that this epithelium is probably one of the “tightest” in the body (Fawcett, 1973).1 The experiments designed to loosen the Sertoli junctions and open the blood-testis barrier indicate that these membrane specializations are also very resistant to dissociation. Exposure to hypertonic solutions ’ Some objection has been voiced to the term “Sertoli junction” on the ground that this specialization might be regarded as simply a variant of the occluding junctions found in many other epithelia. For this reason it was suggested that it might be advisable to use the general term “zonula occludens” with a suitable modifier to take into account the atypical association of the intramembrane particles with the Bface. We prefer “Sertoli junction,” however, because this term serves to emphasize not only the uniqueness of the intramembrane specializations described here but also includes the associated cytoplasmic filaments and cisternae which are equally important as defining characteristics of this complex, and are features not likely to be found in other epithelia.

FIG. 16. Photomicrograph of a seminiferous tubule from a rat testis perfused with hypertonic lithium chloride followed in a few minutes by the aldehyde fixative. The spermatogonia have detached from the basement lamina and shrunken so that the basal compartment of the epithelium appears empty. x 350. FIG. 17. Seminiferous epithelium from the same preparation at higher magnification. It is evident that the basal cytoplasm of the Sertoli cells is condensed and shrunken but the cells remain attached. The spermatocytes and spermatids show no osmotic damage having been protected at these short time intervals by the permeability barrier. x 1200.

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that rapidly open the blood-brain barrier (Rapoport et al., 19741 and dissociate hepatic occluding junctions in a matter of seconds (Goodenough and Gilula, 1974) was ineffective in breaching the Sertoli junctions over a period of several minutes. An unexpected benefit of these experiments was a dramatic visual demonstration of the postulated basal compartment of the epithelium. As a consequence of detachment and extreme shrinkage of the spermatogonia, this compartment appeared in sections as a row of sizeable lacuriae between the basal lamina and the shrunken bases and overarching processes of the Sertoli cells. Also of interest is the striking difference of these two cell types in their relations to the basal lamina. The spermatogonia appear to be only very loosely attached and immediately retract when exposed to hypertonic solutions while the supporting cells remain adherent. Indeed, so firmly are the latter attached that, at longer time intervals, the columnar portion of the Sertoli cells may become so shrunken and attenuated that it gives way leaving an anucleate pyramidal portion of its base still attached to the basal lamina. The differing relationship of the two cell types to their substrate favors the interpretation that the basal lamina may be solely a product of the sessile, supporting cells while the spermatogonia are simply a mobile population residing in expansions of the intercellular clefts of what is basically an epithelium of Sertoli cells. The ontogeny of the seminiferous tubules is consistent with this view in that the gonocytes migrate into the epithelium and first take up positions in the interior of epithelial cords and only secondarily do

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they migrate to the basal lamina at the periphery of the tubule shortly before the onset of spermatogenesis. Spermatogenesis is a precisely ordered, cyclic process in which developmental events are synchronized in clearly defined stages that occupy successive segments along the length of the tubule. Synchrony of differentiation in local areas of the tubule wall is insured by the syncytial nature of germ cell clones which form clusters of up to hundreds of interconnected spermatids (Fawcett, Ito, and Slautterback, 1959; Dym and Fawcett, 1971). Synchrony is not confined to these clusters, however, but extends over segments up to a millimeter long, including many thousands of germ cells (Roosen-Runge, 1962). Thus, an explanation is needed for coordination of events over considerable distances. It has been reported in earlier publications (Fawcett, 1974b, 1975) that gap junctions are not found between supporting cells and germ cells nor between members of neighboring germ cell syncytia. Such junctions are sites of very firm cellto-cell attachment and therefore their occurrence at the interface between the two categories of cells in the seminiferous epithelium would interfere with movement of the germ cells toward the lumen. Thus, the commonest device for communication among epithelial cells seems to have been sacrificed in the seminal epithelium in the interests of maintaining upward mobility of the germ cells. If electrotonic communication does not take place between germ cell clusters or between germ cells and their supporting cells, one may attribute integration of function within the epithelium to the rela-

FIG. 18. Electron micrograph of the base of the seminiferous epithelium after perfusion with hypertonic lithium chloride. The myoid cells are shrunken but intact. The spermatogonium that formerly tilled this niche of the basal compartment is extremely shrunken and retracted. The thin Sertoli cell processes that form the upper boundary of the compartment are shrunken and condensed, while the overlying spermatids in the adluminal compartment are normal in appearance. x 10,000. FIG. 19. Micrograph of a similar area showing an intact Sertoli junction on the boundary between processes overarching the expanded basal compartment. Thus, unlike other epithelial junctions, the Sertoli junctions are resistant to dissociation by hypertonic solutions. x 21,000.

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tions between Sertoli cells. The observation, reported here, of atypical gap junctions on the A-fracture face intercalated between the parallel grooves and discontinuous rows of particles of the Sertoli junctions now provides the most probable structural basis for coordination of the cytological events of the spermatogenic cycle. The observations reported here on the immature and mature seminiferous tubules demonstrate that striking changes occur in junctional membrane specializations during organogenesis and the maturation of the blood-testis barrier. In the immature testis, the barrier is not present, yet the intramembrane specializations between Sertoli cells (Sertoli and gap junctions) are already present and widely distributed within the epithelium. In the course of maturation of the barrier, the junctional specializations are modified and realigned in order to provide an effective barrier and, at the same time, to retain the function of communication. These observations suggest that there are two apparently separate but interdependent processes associated with junctional development during organogenesis: (1) junctional membrane differentiation, and (2) junctional maturation (Gilula, 1973). During these two processes, the junctions may have very different functional properties. In the first process, junctional membrane differentiation, the chemically and structurally differentiated junctional elements are introduced into the membrane where they can be identified as junction-specific. For example, in the immature tubules, the Sertoli junctions can be identified as the nonoriented linear particle aggregates (B face), while the gap junctions are present as numerous polygonal aggregates of particles (A face) that are frequently associ-

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ated with discontinuous segments of the Sertoli junctions. During the second process, junctional maturation, the linear segments of Sertoli junctions are assembled into extensive parallel rows that are aligned horizontal to the axis of the Sertoli cells. Without this final maturation step, the blood-testis barrier is ineffective. Concomitantly, the gap junctions are significantly reduced in number and size, and the junctional particles are arranged into linear aggregates that are usually completely sequestered among the Sertoli junction elements. The linear arrangement of gap junctional particles represents an unusual pleiomorphic form of this communicating junction. Similar junctions have previously been observed in the mammalian retina (Raviola and Gilula, 19731, and gap junctions are frequently present as sequestered elements in other mammalian tissues (Friend and Gilula, 1972). The developmental changes described probably reflect a close association (functional and structural) between the Sertoli and gap junctions, and a reduced requirement for cell-to-cell communication in the mature tissue. It would now be of interest to determine if there are any qualitative or quantitative changes in cell communication between Sertoli cells concomitant with the development of the barrier. A central unsolved problem of modern cell biology is how factors affecting the outside of the membrane or causing perturbations of its internal structure bring about metabolic changes and structural reorganization within the cytoplasm. We have directed our attention in this paper primarily to the occlusive function of the Sertoli junctions as it relates to access of materials to the germ cells from the interstitium. We have not been able to cast any light upon the significance of the associ-

FIG. 20. Photomicrograph of a 13-day postnatal rat testis. At this stage the blood-testis barrier has not been established and the seminiferous cords still lack a lumen. x 1000. FIG. 21. Photomicrograph of 13-day rat testis exposed to hypertonic lithium chloride. In the absence of a blood-testis barrier, the osmotic effects are not confined to the periphery but extend throughout the seminiferous cords. x 1000.

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ated bundles of filaments or the mechanism responsible for their consistent orientation parallel to the rows of intramembranous particles. Also unexplained is the function of the cisternae of the endoplasmic reticulum that invariably course parallel to the specialized zonula of cell-tocell attachment. An adequate interpretation of the Sertoli junctions will have to assign a function to all components of the complex and not merely to the specializations of the membranes. The juxtaluminal zonula occludens of most epithelia is assumed to be a rather stable part of the cell surface, possibly enduring for the interphase lifetime of the cells joined. The occluding Sertoli junctions, on the other hand, cannot endure unchanged for longer than one cycle of the seminiferous epithelium, for it is then necessary for the next generation of germ cells to move from the basal to the adluminal compartment. A major defect in our understanding of the seminiferous epithelium is how this translocation takes place. It could involve transient dissolution of the Sertoli junctions, upward movement of the preleptotene spermatocytes, and reformation of the junctions below them. Alternatively, there may be a progressive dissociation of the particle rows of the broad junction above the germ cells and a concurrent stepwise formation of new rows of attachment between Sertoli cell processes interposed between the germ cells and the basal lamina. By this latter mechanism, the barrier would be maintained at all times during transition of the next generation of germ cells from the basal to the adluminal compartment. Experimental evidence involving use of electron opaque tracers (Dym and Fawcett, 1970; Aoki and Fawcett, 1975) seems consistent with this latter mechanism. If the Sertoli junctions dedifferentiated completely at sites along the length of the tubules where germ cells were in transit, tracers should penetrate to the lumen in these regions. In the absence of any evidence of such local breakdown of

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the Sertoli junctions, one must conclude that there is a gradual upward movement of cells from basal to adluminal compartment without interruption of the permeability barrier. It must be borne in mind that it is not a question of upward movement of single cells but of syncytial chains or clusters of dozens of cells connected by bridges (Dym and Fawcett, 1971; Moens and Hugenholtz, 1975). There is no pseudopod formation or other morphological evidence that the early germ cells are themselves actively motile, and their interconnection would surely interfere with their concerted upward movement. It seems likely therefore that they are passively separated from the basal lamina by undermining processes of the neighboring Sertoli cells. The “zippering up” of the junctions between these processes and the concurrent “unzippering” of the junctions above the germ cells may well be an important part of the mechanism responsible for their ascent into the adluminal compartment. The local control of this process presents a challenging problem to students of spermatogenesis. The development of the Sertoli junctions does not seem to depend directly upon circulating gonadotropic hormones (Vitale et al., 1973). The observation that they develop or at least persist in Busulfan-treated animals indicates that their formation and maintenance does not depend upon inductive interaction of germ cells with their supporting cells. It seems inescapable, however, that their opening and closing at exactly the appropriate stage of the cycle must somehow be dependent upon signals emanating from the germ cells when they reach a certain level of differentiation. Intercellular junctions appear to be essential to the development of nearly all multicellular organisms. Junctions permitting communication between cells are obviously important for coordination of cellular activities. The deployment of occluding junctions for sequestration of

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groups of cells within a compartment where the surrounding cells can create and maintain a special local environment, is emerging as a common device of Nature for promoting differentiation of cells with unusual specific requirements. The isolation of the meiotic and postmeiotic germ cells in the adluminal compartment of the seminiferous epithelium has certain obvious analogies to the early development of occluding junctions in the morula resulting in isolation of the inner cell mass in the special fluid environment of the blastocyst (Ducibella et al., 1974). Note added in proof. While this paper was in press, we have had an opportunity to read the manuscript of a paper by T. Nagano and F. Suzuki: Postnatal development of the junctional complex of mouse Sertoli cells as revealed by freeze-fracture. Anot. Rec., in press. The findings are closely comparable to those reported here. REFERENCES AOKI, A., and FAWCETT, D. W. (1975). Impermeability of Sertoli cell junctions to prolonged exposure to peroxidase. Andrologia 7, 63-76. BARR, L., DEWEY, M. M., and BERGER, W. (1965). Propagation of action potentials and structure of the nexus in cardiac muscle. J. Gen. Physiol. 48, 797. BROKELMAN, J. (1963). Fine structure of germ cells and Sertoli cells during the cycle of the seminiferous epithelium in the rat. Zeitschr. Zellforsch. 59, 820-850. CHALCROFT, J. P., and BULLIVANT, S. (1970). An interpretation of liver cell membrane and junction structure based on observations of freeze-fracture replicas of both sides of the fracture. J. Cell Biol. 47, 49-60. CLAUDE, P., and GOODENOUGH, D. A. (1973). Fracture faces of zonulae occludentes from “tight” and “leaky” epithelia. J. Cell Biol. 58, 390-400. CLERMONT, Y., and PERCY, B. (1957). Quantitative study of the cell population of the seminiferous tubules in immature rats. Amer. J. Anat. 100, 241-267. DREIFUSS, J. J., GIRARDIER, L., and FORSSMANN, W. G. (1966). Etude de la propagation de l’excitation dans le ventricule de rat du moyen de solutions hypertoniques. Pflugers Arch&. 292, 13-33. DUCIBELLA, T., ALBERTINI, D., ANDERSON, E., and BIGGERS, J. (1974). Junctions of the preimplantation mammalian embryo. Characterization and sequential appearance during development. J. Cell Biol. 63, 89a.

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(1973). The fine structure of the monkey Sertoli cell and its role in maintaining the blood-testis barrier. Anat. Rec. 175, 639-656. DYM, M., and FAWCETT, D. W. (19701. The bloodtestis barrier in the rat and the physiological compartmentation of the seminiferous epithelium. Biol. Reprod. 3, 308-326. DYM, M., and FAWCETT, D. W. (1971). Further observations on the numbers of spermatogonia, spermatocytes and spermatids joined by intercellular bridges in mammalian spermatogenesis. Biol. Reprod. 4, 195-215. FARQUHAR, M. G., and PALADE, G. E. (1963). Junctional complexes in various epithelia. J. Cell Biol. 17, 375-412. FAWCETT, D. W. (1973). Interrelations of cell types within the seminiferous epithelium and their implications for control of spermatogenesis. In “The Regulation of Mammalian Reproduction” (Segal, Crozier, Corfman, and Condiffe, eds.), pp. 116135. Charles C Thomas, Springfield, Ill. FAWCETT, D. W. (1974al. Observations on the organization of the interstitial tissue of the testis and on the occluding cell junctions in the seminiferous epithelium. “Schering Symposium on Contraception - Male Gender,” Advances in Biosciences #lo. Pergamon Press, Vieweg. FAWCETT, D. W. (1974133. Interactions between Sertoli cells and germ cells. In “Male Fertility and Sterility” CR. E. Mancini and L. Martini, eds.), pp. 13-36. Academic Press, New York. FAWCETT, D. W. (19751. The ultrastructure and functions of the Sertoli cell. In “Handbook of Physiology,” Vol. 5, Sect. 7: Endocrinology, pp. 21-55. Physiological Society, Washington, D.C. FAWCETT, D. W., ITO, S., and SLALJTTERBACK, D. L. (1959). The occurrence of intercellular bridges in groups of cells exhibiting synchronous differentiation. J. Biophys. Biochem. Cytol. 5, 453-460. FAWCETT, D. W., LEAK, L. V., and HEIDGER, P. M., JR. (1970). Electron microscopic observations on the structural components of the blood-testis barrier. J. Reprod. Fert., Suppl. 10, 105-122. FLICKINGER, C., and FAWCETT, D. W. (19671. The junctional specializations of Sertoli cells in the seminiferous epithelium. Anat. Rec. 158, 207-222. FRIEND, D. S., and GILULA, N. B. (1972). Variations in tight and gap junctions in mammalian tissues. J. Cell Biol. 53, 758-776. GILULA, N. B. (1973). Development of cell junctions. Amer. 2001. 13, 1109-1117. GILULA, N. B. (1974). Junctions between cells. In “Cell Communication” (R. P. Cox, ed.), pp. 1-29. Wiley and Sons, New York. GILULA, N. B., BRANTON, D., and SATIR, P. (1970). The septate junctions: A structural basis for intercellular coupling. Proc. Nat. Acad. Sci. USA 67, 213-220. GILULA, N. B., REEVES, 0. R., and STEINBACH, A. (MUCUCU)

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(1972). Metabolic coupling, ionic coupling, and cell contacts. Nature (London) 235, 262-265. GOODENOUGH, D. A., and GILULA, N. B. (1974). The splitting of hepatocyte gap junctions and zonulae occludentes with hypertonic disaccharides. J. Cell Biol. 61, 575-590. JACKSON, H. (19651. Problems in the chemical control of male fertility. In “Agents Affecting Fertility” (C. R. Austin and J. S. Perry, eds.) pp. 62-77. Churchill, London. MCNUTT, N. S., and WEINSTEIN, R. S. (1973). Membrane ultrastructure at mammalian intercellular junctions. Prog. Biophys. Molec. Biol. 26, 45-101. MOENS, P. B., and HUGENHOLTZ, A. D. (19751. Rat spermatogenesis: A numerical analysis based on quantitative electron microscopy. Biol. Reprod., In press. NICANDER, L. (1967). An electron microscopical study of cell contacts in the seminiferous tubules of some mammals. Zeitschr. Zellforsch. 83, 375397. RAPOPORT, S. I., and THOMPSON, H. K. (1973). Osmotic opening of the blood-brain barrier in the monkey without associated neurological deficits. Science 180, 971. RAPOPORT, S. I., NORI, M., and KLATZO, I. (19711. Reversible osmotic opening of the blood brain barrier. Science 173, 1026-1028. RAVIOLA, E., and GILULA, N. B. (1973). Gap junctions between photoreceptor cells in the vertebrate retina. Proc. Nat. Acad. Sci. USA 70, 16771681.

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ROOSEN-RUNGE, E. C. (1962). The process of spermatogenesis in mammals. Biol. Rev. 37, 343-377. SETCHELL, B. B. (1970). Testicular blood-supply, lymphatic drainage and secretion of fluid. In “The Testis” (A. D. Johnson, W. R. Gomes, N. L. Van Dlemark, eds.), Vol. 1, pp. 101-239. Academic Press, New York. SETCHELL, B. B., VOGLMAYR, J. K., and WAITES, G. M. H. (1969). A blood-testis barrier restricting passage from blood to rete testis fluid but not to lymph. J. Physiol. (London) 200, 73. STAEHELIN, L. A. (1973). Further observations on the fine structure of freeze-cleaved tight junctions. J. Cell Sci. 13, 763-786. STAEHELIN, L. A. (1974). Structure and function of intercellular junctions. Internat. Rev. Cytol. 39, 191-284. STAEHELIN, LIAMS,

L. A., MUKHERJEE, T. M., and WILA. W. (1969). Freeze-etch appearance of tight junctions in the epithelium of small and large intestine of mice. Protoplasma 67, 165-184. STEINBERGER, A., and STEINBERGER, E. (1971). Division pattern of the Sertoli cells in maturing rat testis in vivo and in organ culture. Biol. Reprod.

4, 84-87. 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. Amt. Rec. 176, 333-344. WADE, J., and KARNOVSKY, M. J. (1974). The structure of the zonula occludens. J. Cell Biol. 60, 168180.