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 spermatids 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.     

Actin-like filaments in the Sertoli cell junctional specializations in the swine and mouse testis

Microfilaments at the junctional specializations between adjacent Sertoli cells and between the Sertoli cell and the late spermatid of the mouse and swine testes bind HMM and form arrowhead complexes with a periodicity of about 35 nm. The arrowhead formation is inhibited when the tissues are treated with HMM in the presence of ATP. These observations show that the microfilaments are actin-like in nature. The functional significance of these filaments in the Sertoli cell is discussed.     

Evidence that vinculin is co-distributed with actin bundles in ectoplasmic ("junctional") specializations of mammalian Sertoli cells.

Ectoplasmic specializations of Sertoli cells are actin containing structures found at sites of attachment to spermatids and to neighboring Sertoli cells. We suspect that these cytoskeletal structures are a form of actin-associated adhesion junction. If this is true, then molecular components, such as vinculin, that characterize actin-associated adhesion junctions in general should be present in ectoplasmic specializations. In this paper we have used two approaches to verify the prediction that vinculin is a component of ectoplasmic specializations. First, we have used fluorescence microscopy to probe immunologically for vinculin in ectoplasmic specializations associated with spermatids of the ground squirrel. Second, we have used immunogold techniques to probe for vinculin in ectoplasmic specializations of rat testis. Our results indicate that the immunological probe for vinculin was reactive with ectoplasmic specializations. In single label fluorescence experiments, linear patterns obtained with the vinculin probe were similar to those obtained with probes for filamentous actin. In double label experiments, the vinculin probe was co-distributed with the actin probes. In immunogold studies, specific labelling with the probe for vinculin occurred in ectoplasmic specializations both at sites of attachment to spermatids and adjacent to basal Sertoli cell junctions. Moreover, gold particles were concentrated adjacent to filament bundles within each ectoplasmic specialization. Our results support the conclusion that vinculin is present in ectoplasmic specializations. Further, they indicate that vinculin is co-distributed with actin bundles within each ectoplasmic specialization.     

Ectoplasmic specializations in the Sertoli cell: new vistas based on genetic defects and testicular toxicology

The ectoplasmic specialization is a unique junctional complex formed in two cortical areas of the Sertoli cell in the mammalian testis: one near the base of the seminiferous epithelium forming the blood—testis barrier, and the other near the lumen of the seminiferous tubule embracing the acrosome region of the developing spermatids. The specialization consists of the Sertoli cell plasma membrane, a subsurface cistern of the endoplasmic reticulum and a layer of closely packed actin filaments that is sandwiched between the plasma membrane and the subsurface cistern. No functions of the specializations other than the blood—testis barrier have been established. However, over the past decade, knowledge about the ectoplasmic specialization has been steadily accumulating and, in particular, there has been a tremendous increase in knowledge based on molecular biological approaches to specialization-associated proteins, including tight junction-associated proteins, based on phenotype analyses of gene-knockout mice and mutant animals, and based on analyses of the effects of exogenous estrogens, so-called endocrine disruptors. Progress in studies on the ectoplasmic specialization will facilitate the elucidation of numerous important questions regarding spermatogenesis, including the pathogenesis of azoospermia and the mechanisms of action of endocrine disruptors.     

The Sertoli cell junctional specializations and their relationship to the germinal epithelium as observed after efferent ductule ligation.

Seminiferous tubules from testes of normal and efferent ductule ligated mice were examined with the electron microscope. The tubules in the ligated animals were markedly distended and at most stages of the seminiferous cycle the epithelium exhibited a series of circumferentially-oriented ridges. Cross-sectional profiles of these ridges were studied with particular emphasis on the Sertoli cell junctional specializations and their relationship to the germinal cells. In the ligated specimen the basal cytoplasm of the Sertoli cells is highly attenuated, often appearing as a thin process resting on the basement lamina. Where the cytoplasm of one Sertoli cell ends, it meets in apposition with the cytoplasm of an adjoining Sertoli cell, and at these sites, junctional specializations are present. The ridges are comprised of a stalk of apical Sertoli cell cytoplasm, often appearing like an inverted cone, with young spermatids aligned along the lateral surfaces and the more mature spermatid population embedded within the apical cytoplasm. Junctional specializations were observed along these lateral Sertoli cell surfaces. In some instances, they formed a free surface, but usually early spermatids were in contact with the junctional specializations. With respect to the more mature spermatids, the acrosomal component was typically found in relation to a junctional specialization. Germ cells at the spermatocyte stage were also noted in relation to the Sertoli cell junctional specializations. The findings suggest that spermatocytes cross the Sertoli cell barrier and gain access to the adluminal compartment of the seminiferous tubule through the disengagement of the inter-Sertoli cell junctional complex. It is proposed that when the inter-Sertoli cell junctional specializations separate, the spermatocytes come in apposition with the newly freed junctional surfaces and remain in relation with them through the ensuing divisions. It appears that at some point, firm adhesion between germ cells and the junctional specializations occurs; the spermatid progeny may thus maintain contact with the original inter-Sertoli cell junctional specializations until their release into the tubule lumen.     

Intercellular junctional specializations in human basal cell carcinoma

Summary Intercellular junctions of various types were found on the membrane fracture faces of human nodular basal cell carcinoma (BCC) cells. The junctional types represented include desmosomes, tight junctions, and gap junctions. A semiquantitative comparison of undifferentiated and differentiated nodular BCC showed that gap and tight junctions were observed on all exposed membrane fracture interfaces of the differentiated tumors, while only fifty six per cent of the membrane interfaces of the undifferentiated tumor exhibited similar junctional specializations. These membrane specializations may be a partial reflection of differentiation among the different types of BCC and their contribution to the less invasive character of nodular BCC cannot be ruled out.Preliminary results from this study were presented at the X Triennial World Congress of Pathology Sept. 25–29, 1978, Rio de Janeiro, Brazil     

Development of Sertoli cell junctional specializations and the distribution of the tight-junction-associated protein ZO-1 in the mouse testis

Basally located tight junctions between Sertoli cells in the postpubertal testis are the largest and most complex junctional complexes known. They form at puberty and are thought to be the major structural component of the “blood–testis” barrier. We have now examined the development of these structures in the immature mouse testis in conjunction with immunolocalization of the tight-junction-associated proteins ZO-1 (zonula occludens 1). In testes from 5-day-old mice, tight junctional complexes are absent and ZO-1 is distributed generally over the apicolateral, but not basal, Sertoli cell membrane. As cytoskeletal and reticular elements characteristic of the mature junction are recruited to the developing junctions, between 7 and 14 days. ZO-1 becomes progressively restricted to tight junctional regions. Immunogold labeling of ZO-1 on Sertoli cell plasma membrane preparations revealed specific localization to the cytoplasmic surface of tight junctional regions. In the mature animal, ZO-1 is similarly associated with tight junctional complexes in the basal aspects of the epithelium. In addition, it is also localized to Sertoli cell ectoplasmic specializations adjacent to early elongating, but not late, spermatids just prior to sperm release. Although these structures are not tight junctions, they do have a similar cytoskeletal arrangement, suggesting that ZO-1 interacts with the submembrane cytoskeleton. These results show that, in the immature mouse testis, ZO-1 is present on the Sertoli cell plasma membrane in the absence of recognizable tight junctions. In the presence of tight junctions however, ZO-1 is found only at the sites of junctional specializations associated with tight junctions and with elongating spermatids.     

Granular transformation of Sertoli cells in testicular disorders.

In order to study the granular transformation of Sertoli cells the following testicular specimens were reviewed: 58 postmortem biopsies from 21 children and 37 young adult males with normal histologic pattern; 165 biopsies from prepubertal cryptorchid testes; 38 biopsies and 18 surgical specimens from postpubertal-cryptorchid testes; bilateral biopsies from eight men with Del Castillo's syndrome, 14 men with retractile testes, and five men with obstructive azospermia; 17 bilateral and seven unilateral biopsies from 24 men with varicocele; seven unilateral biopsies plus five surgical specimens from 12 men with male pseudohermaphroditism; one biopsy and one surgical specimen from two men with macroorchidism; and the autopsy specimens from 28 adult men with acquired immunodeficiency syndrome (AIDS). Sertoli cells with eosinophilic granular cytoplasm were found in the testes of one prepubertal and four postpubertal cryptorchid males, two males with Del Castillo's syndrome, two males with retractile testes, four males with varicocele, two male pseudohermaphrodites, two males with macroorchidism, and one male with AIDS and interstitial orchitis. Histochemical and ultrastructural examination of granular Sertoli cells revealed that these cells accumulate secondary lysosomes and show scant cytoplasmic organelles. In the males with varicocele or retractile testes, these lysosomes were probably heterolysosomes that had degraded the germ cells and testicular fluid accumulated in the lumen of the ectatic seminiferous tubules of these testes. A similar mechanism is also probable in the male with interstitial orchitis that had caused germ cell destruction. In the other cases, in which the tubules showed reduced lumen and severe germ cell depletion, the abundant lysosomes are probably cytolysosomes. The development of these cytolysosomes might be related to the Sertoli cell dysgenesis present in these testes.     

Late metastasis after a testicular Sertoli cell tumour

The diagnosis of Sertoli cell tumors is sometimes difficult and can be improved using anti inhibin immunohistochemistry. It is also difficult to establish the prognostic of Sertoli cell tumors. In our observation a high Mib 1 rate (20%) could have be taken in account to decide a better survey or/and a lymphadenectomy, which could have avoided lymph node metastasis in our patient, which was discovered ten years after orchidectomy.     

The Sertoli cell junctional specialization during spermiogenesis and at spermiation

The relationship between developing spermatids and Sertoli cell junctional specializations was studied with the electron microscope during spermiogenesis and at spermiation. At stage I of the seminiferous cycle, the newly formed spermatids are found in apposition to junctional specializations at the lateral surfaces of the Sertoli cell. Visualization of the junctional site of this early stage appears to be dependent on orientation and plane of section. As differentiation proceeds, the spermatids elongate and come to lie within deep recesses of the Sertoli cell. At this time the junctional specialization is limited to the acrosomal portion of the spermatid. During the maturation phase, the spermatids, while maintaining the same relationship to the junctional specialization, approach the lumen. When stage VIII of the cycle is reached, the stage in which spermiation occurs, the spermatids are at the luminal surface. The relationship of the spermatid head to the junctional specializations is quite variable during this stage. Some spermatids are observed still attached to the Sertoli cell at the junctional site, while others are found completely or partially surrounded by Sertoli cytoplasm, but with no evidence of the normally interposed junctional specialization. Yet, in other instances, the spermatids are observed in a position slightly removed from the junctional site. Also evident are profiles of junctional specializations at a free surface of the Sertoli cell, there being no attached spermatid. In some instances the junctional specializations appeared in apposition to a residual body. In the case of the free surface profiles, the junctional specialization at times lined an empty cleft or crypt-like recess, giving the impression that the spermatid head had just been dislodged from the junctional contact site. The findings indicate that the spermatid is in contact with a junctional specialization from its initial appearance and remains so until spermiation is initiated. It is postulated that spermiation is initiated through a physiological change in the junctional specialization resulting in loss of adhesion and consequent release of the sperm head from its attachment site. A similar mechanism is proposed in relation to the inter-Sertoli junctional complex to account for the means by which the spermatocytes cross this barrier to reach the adluminal compartment of the seminiferous epithelium.     

Spermiogenesis in Nautilus pompilius. II. Sertoli cell-spermatid junctional complexes

A "ball and socket-like" junction between branches of the Sertoli cells and the developing spermatids is described. A cytoplasmic extension of the Sertoli cell fits into a pocket in the spermatid and has a constricted neck region. Frequently there are large multivesiculate bodies in the Sertoli cell extension and small vesicles frequently appear in the spermatid cytoplasm, in the area of the "ball and socket-like" junction. This suggests the possibility that there may be communication of materials between the two cells. The possible function of the junctions is discussed and it is concluded that they more likely have a role in nutrition than in coordination.     

Chlorphentermine-induced lipidosislike ultrastructural alterations in testicular Leydig and Sertoli cells

Prolonged administration of the anorectic drug chlorphentermine to rats, mice, and rabbits causes the formation of abnormal cytoplasmic inclusions in Leydig and Sertoli cells. The abnormal inclusions display either concentrically lamellated patterns with a 45 Å periodicity, or a crystalloid structure. These alterations correspond to chlorphentermine-induced changes previously observed in other tissues; they are interpreted as the result of a drug-induced phospholipidosis.     

Maturation phenotype of Sertoli cells in testicular biopsies of azoospermic men

The involvement of Sertoli cells in different spermatogenic impairments has been studied by an immunohistomorphometric technique using cytokeratin-18 (CK-18) as a marker for immature Sertoli cells. CK-18 is known to be expressed in Sertoli cells during prenatal and prepubertal differentiation and is normally lost at puberty. Forty-nine azoospermic men were included in the current study. Quantitative measurements on testicular biopsies revealed the highest CK-18 expression in the mixed atrophy biopsies (22 men), a lower expression in the Sertoli cell-only (SCO) biopsies (12 men), and minimal residual staining in the group considered as representing normal spermatogenesis (six obstructive azoospermia patients). The cytokeratin immunopositive-stained tubules were associated either with arrest in spermatogenesis or with SCO. Examination of sections from nine men with microdeletions in the AZF region of the Y chromosome revealed that these men were either negative for CK-18 expression or showed only weak residual staining. This may suggest that the spermatogenic defect in the AZF-deleted men originates in the germ cell and has no impact on Sertoli cell maturation. The cause that determined the spermatogenic defect in the other cases of male infertility with high CK-18 expression may have damaged both the Sertoli and the germ cells.     

Distribution of actin in Sertoli cell ectoplasmic specializations and associated spermatids in the ground squirrel testis

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

Mitotic activity of Sertoli cells in adult human testis: an immunohistochemical study to characterize Sertoli cells in testicular cords from patients showing testicular dysgenesis syndrome

During puberty, normal somatic Sertoli cells undergo dramatic morphological changes due to the differentiation of immature pre-Sertoli cells in functionally active adult Sertoli cells. Sertoli cell maturation is accompanied with loss of their mitotic activity before onset of spermatogenesis and loss of pre-pubertal and occurrence of adult immunohistochemical Sertoli cell differentiation markers. Testes of infertile adult patients often exhibit numerous histological signs of testicular dysgenesis syndrome (TDS) such as microliths, Sertoli cell only (SCO) tubules, tubules containing carcinoma in situ and immature seminiferous tubules (Sertoli cell nodules). Sertoli cell tumours, however, are very rare neoplasms possibly due to the fact that the mechanism and temporal origin of neoplastic Sertoli cells underlying Sertoli cell tumourigenesis still remain unknown. To clarify the state of Sertoli cell differentiation in both immature seminiferous tubules of adult patients with TDS and Sertoli cell tumour, we compared the expression of the Sertoli cell differentiation markers vimentin, inhibin-α, anti-Muellerian-hormone, cytokeratin 18, M2A-antigen, androgen receptor and connexin43 with that of SCO tubules with hyperplasia. In addition, we demonstrated for the first time the existence of proliferating Sertoli cells by Ki67- and PCNA-immunostaining in Sertoli cell nodules of the adult human testis. Our data indicate that mitotically active Sertoli cells in Sertoli cell nodules will be arrested prior to puberty and, contrary to dogma, do not represent foetal or neonatal cells. Since all markers in Sertoli cell nodules revealed a staining pattern identical to that in neoplastic Sertoli cells, but different to that in Sertoli cells of SCO tubules with hyperplasia, it may be speculated that Sertoli cell tumours in adult men may originate from Sertoli cell nodules.     

Hyperplasia and the immature appearance of Sertoli cells in primary testicular disorders

Testicular biopsy specimens from adult patients affected with cryptorchidism, Klinefelter's syndrome, and Del Castillo's syndrome were examined by light and electron microscopy. The study revealed a high proportion of testes showing seminiferous tubules with hyperplasia of Sertoli cells (from 25 to 45 cells per transverse tubular section). These cells had an immature appearance and showed a pseudostratified distribution. The nucleus was round to ovoid and regular in outline, with a smaller nucleolus than that of mature Sertoli cells. The cytoplasm showed less development of the endoplasmic reticulum as well as of the secondary lysosomes and lipid droplets than that in mature Sertoli cells. Characteristic features of these immature Sertoli cells were abundant cytoplasmic microfilaments, elaborate interdigitations between adjacent cells, and extensive tight junctions, from basement membrane to lumen. In the cryptorchid testes, a more immature Sertoli cell was found to constitute the majority of the cells in hypoplastic zones. In Klinefelter's and Del Castillo's syndromes as well as in cryptorchid testes to a lesser degree, a transitional type of cell-from immature to mature-was also observed. These observations suggest that Sertoli cells in these primary testicular disorders reflect a congenital deficiency producing abnormal development.     

Morphometric and cytogenetic characteristics of testicular germ cells and Sertoli cell secretory function in men with non-mosaic Klinefelter's syndrome

BACKGROUND: Klinefelter's syndrome is the most frequent chromosomal abnormality in infertile men. In this study, the chromosomes of round spermatids and spermatogonia/primary spermatocytes from men with non-mosaic Klinefelter's syndrome were examined, together with the Sertoli cell secretory function and sperm morphometry. METHODS: Twenty-four men with non-mosaic Klinefelter's syndrome and nine men with obstructive azoospermia underwent therapeutic testicular biopsy. When spermatozoa in the final filtrate were present, they were processed for sperm morphometry or ICSI. Sperm morphometry was evaluated by the maximal length and width of the sperm head, the length of the midpiece and the ratio of the acrosomal region to the total surface area of the head. When round spermatids were present, they were processed for fluorescent in-situ hybridization (FISH). FISH was also applied to fragments of seminiferous tubules. Sertoli cell secretory function was measured by the amount of androgen binding protein (ABP) secreted in vitro. RESULTS: More than 93% of the evaluated round spermatids were normal. The proportions of 24,XY and of 24,XX round spermatids to the total number were significantly larger in men with Klinefelter's syndrome than in obstructed azoospermic men. Men with Klinefelter's syndrome who had spermatozoa in their testicular tissue (n = 12) were positive for both 46,XY and 47,XXY spermatogonia in their seminiferous tubules. In contrast, men with Klinefelter's syndrome without spermatozoa in their testicular tissue (n = 12) were positive for 47,XXY spermatogonia but negative for 46,XY spermatogonia in their seminiferous tubules. ABP profiles were significantly smaller in men with Klinefelter's syndrome who were negative for spermatozoa compared with men who were positive. Four pregnancies were achieved and five healthy babies were born. CONCLUSIONS: This study suggests that few 46,XXY spermatogonia undergo meiosis with an XX pairing and a Y univalent type of pairing. Hyperhaploid round spermatids (24,XY and 24,XX) may be produced by meiosis of 47,XXY spermatogonia. Men with Klinefelter's syndrome who are negative for testicular spermatozoa have a greater degree of Sertoli cell secretory dysfunction compared with men with Klinefelter's syndrome who are positive for spermatozoa. There are several defects in sperm morphometry with functional significance in men with Klinefelter's syndrome.     

Characterization of Porphyromonas gingivalis-induced degradation of epithelial cell junctional complexes

Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.