Sertoli Cell Junctions Research Articles

Plakoglobin: A protein common to different kinds of intercellular adhering junctions

We have established, by means of a monoclonal antibody and a cDNA clone, that a desmosomal polypeptide of M r 83,000 also occurs at the plaques of other types of adhering junctions, including the vinculin-actin-associated intercellular junctions, e.g., the zonula adhaerens of epithelial cells and the endothelial, lens, and Sertoli cell junctions. This is the first component found in common among otherwise biochemically distinct plaque domains. Despite its concentration at these intercellular junctions, it is absent from the respective cell-substratum contact sites. In addition, it appears in a globular soluble 7S form in the cytoplasm. We discuss the significance of this protein, for which the name plakoglobin is proposed, in terms of its interaction with such biochemically diverse membrane domains and their different types of associated cytoskeletal filaments.

Sertoli cell junctional complexes in gossypol-treated neonatal and adult guinea pigs.

The effect of gossypol, an experimental male contraceptive agent, on the development and maintenance of the blood-testis barrier was determined by feeding gossypol daily to prepubertal and adult guinea pigs, and then examining their testes by electron microscopy of thin sections and freeze-fracture replicas. In guinea pigs of 10 to 30 and 10 to 40 days of age that were fed gossypol, impermeable continuous junctional zones did not develop between adjacent Sertoli cells. Compartmentalization of germ cells in the seminiferous epithelium, therefore, was nonexistent. These findings were obtained by use of the sterol-binding polyene, filipin, used as a low molecular weight tracer in combination with freeze-fracture. In general, the seminiferous tubules lacked lumina and spermatogenesis did not progress beyond the pachytene spermatocyte stage. In adult guinea pigs fed gossypol daily for five weeks, continuous zonules at the base of the seminiferous epithelium appeared intact and were impermeable to filipin. Discontinuous zonules found higher in the epithelium showed distensions between interrupted junctional strands and were permeated by filipin. In addition, vacuolated spaces between Sertoli cells and clumps of heterochromatin were conspicuous in some of the Sertoli cell nuclei. Spermatogenesis was disturbed in about 10% of the seminiferous tubules examined. These perturbations included exfoliation of round and elongated spermatids with concomitant formation of multi-nucleated giant cells. Spermatozoa from these adult male guinea pigs were immotile. These findings suggest that, in neonatal animals, gossypol appears to prevent the maturation of Sertoli cells and this effect is expressed as the failure of focal Sertoli cell tight junctional strands to assemble into continuous zonules. In adult animals, gossypol appears to have no effect on the maintenance of the blood-testis barrier.

Sertoli Cell Junctions: Morphological and Functional Correlates

Publisher Summary Modern cell biology has shown that cellular recognition and adhesion are not always manifested by junctions of a classical type or by morphological entities recognizable by standard transmission electron microscopy techniques. This chapter describes several junctions in which experimental data are the primary evidence for suggesting a junctional relationship between cells. The cyclic nature of spermatogenesis and the continually changing morphology of the Sertoli cell provide a dynamic framework in which junctions are periodically formed and eliminated. Desmosomes are important in maintaining the integrity of the seminiferous epithelium, a function, which should not be underemphasized, because many germ cell types appear only loosely arranged in the seminiferous epithelium. Based on circumstantial evidence, it is reasonable to suspect that, as yet, unrecognized junctional forms are present in the relationship of the Sertoli cell and germ cells and will be revealed in the future through methodological advances.

The blood-testis barrier in the toad (Bufo arenarum Hensel): a freeze-fracture and lanthanum tracer study.

Intercellular junctions between Sertoli cells in the toad testis were studied by freeze-fracture and electron-opaque intercellular markers. These junctional specializations are characterized in thin sections by a series of focal fusions on the outer leaflets of both adjacent cell plasmalemmas, associated with bundles of fine filaments in the subjacent Sertoli cell cytoplasms. However, the wide subsurface cisterna of the endoplasmic reticulum, a component constantly associated with Sertoli cell junctions in mammals, is absent in the toad. The intravascularly injected lanthanum hydroxide, used as a tracer compound, gains access to the seminiferous tubules and surrounds spermatogonia and leptotene spermatocytes, but is persistently excluded from germ cells in later stages of development. This indicates that, as is the case in the mammalian testis, a permeability barrier to lanthanum is established which isolates all germ cells beyond leptotene spermatocytes. Freeze-fracture reveals the characteristic occluding junctions between Sertoli cells, but a variation in their geometric patterns was clearly observed in different regions of the toad seminiferous epithelium. The membrane-fractured faces of Sertoli cells embracing differentiating spermatids exhibit a deep junctional complex: up to 50 rows of particles between adjacent Sertoli cells separate these late germ cells from the periphery of the seminiferous tubules. Sertoli cells surrounding early germ cells generally exhibit, instead, a discontinuous, poorly developed network of interconnected rows of particles with few widely spaced strands. This seems to permit the percolation of the intercellular marker in areas of the seminiferous epithelium containing spermatogonia and leptotene spermatocytes.

The effects of follicle-stimulating hormone (FSH) on sertoli cell junctions in vitro: A freeze-fracture study

The mechanism(s) for the control of Sertoli cell tight junction development and orientation has not been established. We have studied the effect of FSH (5 U/ml) on tight junction formation using an organ culture technique that allows maintenance of tubular structure and viability of Sertoli cells, without the other influencing factors that are encountered in vivo . Tight junctions developed as the control organ cultures aged and consisted of extensive junctional complexes by 30–40 days in culture. Yet their development did not correspond to that seen in the maturing animal at 30 days, remaining basically unoriented. In the 30- to 40-day FSH-treated cultures the tight junctions were not only aligned parallel to one another, but were also condensed in bands at the basal part of the Sertoli cell and seemed to be oriented parallel to the myoid cell layer. These observations suggest that FSH affects the tight junctions of Sertoli cells by influencing their overall orientation. Additionally, the occurrence and possible significance of a septate-like character of the Sertoli junctions that was occasionally noted is discussed.

Fine structure of the Sertoli cells of the rat testis in experimental unilateral cryptorchidism.

Mature rats were made unilaterally cryptorchid for 28 days. ABP concentrations of the abdominal epididymis were significantly reduced, indicating impaired secretory activity of the Sertoli cells. The FSH serum level was increased and testosterone levels both of the serum and of homogenates of the cryptorchid testis were slightly reduced. Sertoli cells of the cryptorchid testis showed signs of regression. Although Sertoli cell junctions were still impermeable to lanthanum, a reduction of membrane particles forming the Sertoli cell junctions was observed in freeze-fracture replicas. In thin sections it was seen that the plasma membrane-apposed cisternae of endoplasmic reticulum or the adjaacent microfilaments were partially lacking. No such changes were observed in the scrotal testis.

Ultrastructural changes of the intercellular relationship in impaired human spermatogenesis.

In seven hypo- or aspermic patients, electron microscopic investigations of the intercellular connections of the seminiferous tubule were performed. The analysis of cell junctions of Sertoli cells and germ cells revealed irregularities of the Sertoli-cell junctions, hypoplasias of occluding junctions, hypo- and hyperplasias of the Sertoli-spermatid cell junctions and abnormal formation of Sertoli cell junctions with early spermatids, spermatocytes, and spermatogonia. Gap junction-like cell membrane specializations were very rare. Intercellular cytoplasmic bridges of germ cells were always present together with these cells. One hypoplastic bridge connecting two spermatogonia was found. The results allow a preliminary classification of impaired spermtogenesia. The changes of intercellular connections might disturb the blood-testis barrier as well as the intercellular communication in the seminiferous tubule. Evidence is available to support the suggestion that genetic causes play a considerable role in the etiology of the germ cell aplasia and the spermatogenic maturation arrest.

Freeze-fracture Observations on the Rat Sertoli Cell Junctions by Metal Contact Freezing

Freeze-fracture Observations on the Rat Sertoli Cell Junctions by Metal Contact Freezing

Hormonal regulation of Sertoli cell differentiation.

This study was carried out to determine the possible hormone dependence of the postnatal changes on the cytological characteristics of the rat Sertoli cell. Neutralizing antisera to rat follicle stimulating hormone (AS/FSH) and sheep luteinizing hormone (AS/LH) were prepared in rabbits and made monospecific by suitable absorption. Groups of male Sprague-Dawley rats were injected from birth to 32 days with either AS/FSH, AS/LH, AS/LH + testosterone propionate (TP), or normal rabbit serum (NRS). In control 33-day-old animals, Sertoli cells displayed most of the fine structural characteristics of the adult type. Their nuclei were highly infolded and showed an elaborate shape. Euchromatin was largely predominant with a few heterochromatic masses associated with the nuclear envelope or with the prominent nucleolus. The perinuclear area of cytoplasm was particularly rich in mitochondria, smooth endoplasmic reticulum (SER) and Golgi complexes. Inter-Sertoli cell junctions were fully developed. After AS/LH treatment, the nuclei were reduced in size and adopted a smooth surface with very few infoldings. In addition, many clumps of heterochromatin were apparent close to the nuclear envelope and the number of the nucleoli was reduced. Sertoli cell cytoplasm was severely atrophied following AS/HL administration, showing sparse mitochondria, SER and Golgi profiles. Addition of TP to the AS/LH treatment failed to reverse these cytoplasmic changes but resulted in a reappearance of the membrane infoldings of the nuclear envelope and normal numbers of nucleoli. On the other hand, AS/FSH treatment did not induce any apparent morphological change in the Sertoli cell nuclei. The cell volume was reduced but most of the cytoplasmic organelles were apparently present in normal numbers. However, extensively dilated cisternae of the endoplasmic reticulum containing floccular material were a common and impressive change. No modifications were detected in the Sertoli cell junctions in any of the experimental groups. ABP testicular and epididymal levels were diminished in both AS/FSH and AS/LH treated groups, but the reduction was more marked in the latter. ABP levels remained low when TP was administered with the AS/LH. These observations indicate that in the absence of LH morphological differentiation and growth of Sertoli cells are severely inhibited and that FSH withdrawal causes specific changes in the ER that may indicate a trophic action on it. The failure of TP to reverse the effects of AS/LH administration could be attributed to the possibly severe FSH inhibition induced by the high testosterone plasma levels.

Sertoli Cell Differentiation in the Testes of Mice Genetically Deficient in Germ Cells

Mutations rendering mice deficient in germ cells provide a natural experimental system for investigation of the differentiation of Sertoli cells without the normal number of germ cells. In the present study, use was made of 2 viable mutants (atrichosis, at/at, and dominant spotting, W/Wv) with testes grossly deficient in germ cells because of the failure of the genital ridges to become adequately populated with germ cells. Ultrastructural examination of prepuberal testes of such mice revealed that essentially normal, both structural and temporal, maturation of the Sertoli cells occurred. In particular, there was normal differentiation of the typical tripartite nucleolus, the Sertoli cell junctions and mitochondria. We conclude that the normal complement of differentiating germ cells is not a prerequisite for structural differentiation of Sertoli cells.

Characterization of Sertoli cell‐germ cell junctional specializations in dissociated testicular cells

To further characterize Sertoli cell-germ cell junctional specializations seminiferous tubules from sexually mature Sprague-Dawley rats were dissociated by enzymatic and mechanical methods. Ultrastructural analysis of cell suspensions prepared by incubation in collagenase alone or by mechanical methods revealed that spermatids remained attached to Sertoli cells or Sertoli cell fragments. Such cellular associations were found only between Sertoli cell fragments and spematids in which the developing acrosome had made contact with the plasma membrane (step 8 and subsequent steps of spermiogenesis). Furthermore, the fragments were confined to that region of the plasma membrane over the acrosome. The Sertoli cell half of this adhesive site displayed the typical elements of Sertoli cell junctions, filamentous bundles and associated cisterna of endoplasmic reticulum, in apposition to the spermatids. The spermatids demonstrated no surface specializations at the attachment sites. In contrast, in cell suspensions prepared with trypsin, spermatids were free of attachments to Sertoli cells or their fragments. These results demonstrate that: (1) the junctions act to bind cells together, (2) adhesive type contact is established between Sertoli cells and spermatids at step 8 and subsequent steps of spermiogenesis, (3) contact is restricted to the spermatid plasma membrane over the acrosome, and (4) spermatids can be freed from the junctional specializations by treatment with trypsin.

Development of Sertoli cell junctions in vitro--a freeze-fracture study.

Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo.

Freeze-etch observations on the tight junctions of sertoli cells grown in organ culture with FSH

The Sertoli cell tight junctional complexes have been shown to be the most important structural counterpart of the physiological blood-testis barrier. In freeze etch replicas they consist of extensive rows of intramembranous particles which are not only oriented parallel to one another, but to the myoid layer as well. Thus the occluding complex has both an internal and an overall orientation. However, this overall orientation to the myoid layer does not seem to be necessary to its barrier function. The 20 day old rat has extensive parallel tight junctions which are not oriented with respect to the myoid layer, and yet they are inpenetrable by lanthanum. The mechanism(s) for the control of Sertoli cell junction development and orientation has not been established, although such factors as the presence or absence of germ cells, and/or hormones, especially FSH have been implicated.

Regional differentiation of cell junctions in the excurrent duct epithelium of the rat testis as revealed by freeze-fracture.

AbstractIntercellular junctions of the excurrent duct system of the adult rat testis were studied by freeze‐fracture. In the terminal segment of the seminiferous tubule, where there are no spermatogenic cells, the tight junctions of Sertoli cells consist of many parallel strands of particles. The particles of the junctions predominantly appear on the E face instead of the P face, similar to those of the seminiferous tubules reported previously. From the rete testis to the ductus deferens, the tight junctional particles or smooth strands are mainly found on the P face, and the tight junctions show anastomosing networks. In the ductuli efferentes, whose epithelial lining consists of ciliated and nonciliated cells, the tight junctions between two adjacent nonciliated cells and between nonciliated and ciliated cells are poorly developed. In the former, belt‐like gap junctions are often associated with segmented tight junctions. In such area, there are tiny regions, where no junctional elements are observed. Between two ciliated cells, several strands of the tight junctions can be seen. The result of the tracer experiment suggests that the barrier of the ductuli efferentes is weak. In the epididymis, the tight junctions are well developed throughout the duct. Corresponding with the regional variation of the epi‐didymal epithelium, the geometrical organization of the tight junction networks varies along the duct. In the ductus deferens, many strands of the tight junctions are scattered throughout the lateral cell surface in addition to the belt‐like network of the tight junction in the adluminal area.The number of the tight junctional strands is presented graphically in the various segments of the excurrent duct of the testis.

Cell to cell relationships in the seminiferous epithelium in the mouse embryo.

Germ cells and Sertoli cells in embryonic mouse testes (day 14 to 20 of gestation) were examined by sectioning and freeze-fracture. Intercellular cytoplasmic bridges between the germ cells are observed in day 14 and older embryos. Membrane specializations with dense fuzzy material similar to the so-called "desmosome-like structures" are found between Sertoli cells and germ cells. A cell contact area with dense opposed membranes is also found between adjacent germ cells. Asymmetrical dense fuzzy lining of both Sertoli and germ cell membranes is noted. Pinocytotic pits or caveolae are frequently found in the Sertoli cell membrane. Between adjacent Sertoli cells, gap junctions of various sizes and focal meshworks of the occluding junctions are found. Most of the occluding junctional particles are located in the center of the grooves in the E face, and are similar to those in postnatal and adult Sertoli cell junctions. In addition, on both fractured faces there are ridges and grooves devoid of particles which are continuous with occluding junctions with particles, suggesting an initial stage in the formation of occluding junctions of the Sertoli cells. Particles gathered at the site of desmosome-like structures are present on the P face of the Sertoli cell.

Ultrastructural details of Sertoli cell junctional complexes in vivo and their modifications in tissue culture.

This study was carried out using thin sections, lanthanum tracing, and freeze-cleaving in order to investigate the Sertoli cell junctions of rat testis in vivo and their maintenance in culture. The presence of a new type of membrane specialization has been revealed. This consists of a close association between tight junctions and nexuses. The Sertoli cell contact specializations show a progressive disorganization in vitro correlated with the duration of the period in culture. The relationship between the morphological changes in Sertoli cell junctions and the lack of spermatogenesis in culture is discussed.

Freeze-fracture observations on the intercellular junctions of Sertoli cells and of Leydig cells in the human testis.

Normal and feminized human testes were examined by means of freeze-fracture. In both cases, the junctional complexes between adjacent Sertoli cells show a unique characteristic feature. Many parallel linear occluding junctions are located circumferentially around the cell surface at the level of the nucleus. They are more than forty in number over one Sertoli cell surface. In the A face, the particles are not prominent on the ridge of the junctions. Instead, they are clearly seen in the center of the groove of the junctions on the B face. Gap junctions consisting of particle aggregation are not found between the Sertoli cells. The gap junctions between the Leydig cells are frequently observed in both normal and feminized testes. They are round or elliptic in contour and about several micrometers in diameter. The particles, about 8 nm in diameter, are closely packed to form a quasi-hexagonal pattern. The pits are found on the B face corresponding to the particle aggregation.

The normal development of the blood-testis barrier and the effects of clomiphene and estrogen treatment.

AbstractPrevious work with electron opaque intercellular markers has indicated that in adults occluding junctions between adjacent Sertoli cells are the principal morphological basis of the blood‐testis barrier (Dym and Fawcett, '70). Since the barrier of not present at birth this study was undertaken to determine when it is established during postnatal development of the rat and to correlate its appearance with other developmental events in the seminiferous epithelium. From birth to 16 days of age interstitially injected tracers, such as horseradish peroxidase, freely entered the seminiferous epithelium and permeated the 200 Å intercellular clefts between presumptive Sertoli cells and germ cells, reaching the center of the seminiferous cords and site of the future tubule lumen. Between day 16 and 19 occluding junctions between Sertoli cells appear and thenceforth interstitially injected electron opaque tracers were effectively prevented from reaching the tubule lumen. Therefore, in rats, the blood‐testis barrier is established between 16 and 19 days of age.In an attempt to determine whether the development of the Sertoli cell junctions and the blood‐testis barrier is under hormonal control circulating gonadotropins were suppressed using daily injections of clomiphene or estrogens from birth. The appearance of blood‐testis barrier was delayed approximately seven days in the absence of gonadotropins. However, by day 26 the Sertoli cell junctions did appear and interstitially injected tracers were prevented from reaching the tubule lumen. Thus the development of the occluding junctions between Sertoli cells that constitute the permeability barrier does not appear to be directly dependent upon gonadotropins.

The local control of mammalian spermatogenesis.

Investigations of the ultrastructure of mammalian spermatogenesis have revealed intercellular bridges in all cell generations of the germinal epithelium. Desmosomes connect Sertoli cells with associated Sertoli cells (Sertoli cell — Sertoli cell junction) and with adjacent cells of the germinal epithelium. To provide evidence for the different types of cell connections in all developmental stages of spermatogenesis the various cell generations had to be worked out. This was achieved by considering the morphology of the nuclei and the relation of the cells to cell associations of the spermatogenic cycle in which they have been observed. These observations led us to a hypothesis explaining the local control of the spermatogenic cycle. According to this hypothesis all cells of one developmental stage within a given cell association are interconnected by intercellular bridges, thus forming a syncytium which contains each complete layer of a cell association. This explains the synchronous development of the cell layers. The coordination of the development of different layers of one cell association is effected by Sertoli cells. To achieve this organization of the spermatogenic cycle the entire cell population of one cell association has to be derived from one common embryonic stem cell. The evidence for this hypothesis is discussed.