Interstitial compartment pathology and spermatogenic disruption in testes from impotent diabetic men

Studies utilizing animal models of diabetes suggest that diabetic complications of impotence involve structural lesions in the testis as part of an overall defect in the pituitary-testicular axis. In the present study testicular biopsies from ten oligospermic and/or impotent men with diabetes were evaluated by light and electron microscopy. One biopsy was judged normal. The remaining tissue showed variable testicular pathology ranging from minimally to grossly affected. Seminiferous tubules had decreased tubule diameters, hyalinized tubule walls, and occluded lumina owing either to epithelial encroachment or cellular debris and exfoliated round germ cells. Sertoli cells were vacuolated and showed a high degree of apical cell membrane redundancy and degeneration. Although Sertoli-Sertoli cell junctional complexes appeared normal, Sertoli junctional specializations associated with spermatids were structurally abnormal or absent. All tubules were variably depleted of adluminal compartment germ cell types. The interstitial compartment was filled with a collagen-rich extracellular matrix concentrated around small blood vessels and seminiferous tubule walls. Capillaries and lymphatic endothelia appeared structurally abnormal and compromised by the interstitial "matrix expansion." Some Leydig cells contained a variable number of small to large lipid droplets, vacuoles, and secondary lysosomes. Results indicate the presence of tissue pathology in testes of impotent diabetic men. Discrete ultrastructural lesions in apical Sertoli cell cytoplasm are associated with spermatogenic disruption and morphological changes in the interstitial compartment suggest microvascular complications.     

Three-dimensional reconstruction of a rat stage V Sertoli cell: II. Morphometry of Sertoli–Sertoli and Sertoli–germ-cell relationships

Sertoli–Sertoli and Sertoli–germ-cell configurational relationships were studied using morphometric techniques and direct measurements as obtained from micrographs used to reconstruct a model of a rat stage V Sertoli cell. Regional areas of the Sertoli cell surface, which faced germ cells, other Sertoli cells, or noncellular structures, were expressed as relative surface area percentages; and the absolute surface areas for these regional areas were calculated. The surface area of the reconstructed cell, in its unmagnified state, was found to be 12,163 μm². Cell processes were enumerated and studied using morphometric techniques. The surface area of the reconstructed Sertoli cell facing germ cells and Sertoli cells was also determined. Five Sertoli cells showed extensive contact with the reconstructed cell at the level of the Sertoli-Sertoli junctional contact region. This contact region averaged 3.51 μm in width. The relative and absolute surface area of subsurface ectoplasmic specialization of the Sertoli cell that faced germ cells and other Sertoli cells was calculated, and the extent of penetration of step 17 spermatids into the Sertoli crypts was determined. Surface relationships of the reconstructed cell to cellular and noncellular elements were depicted on outline drawings of the Sertoli cell.     

Altered expression of connexins 26 and 43 in Sertoli cells in seminiferous tubules infiltrated with carcinoma-in-situ or seminoma

The expression of connexins (cx) 26 and 43 in testis infiltrated with carcinoma-in-situ (CIS) or seminoma was examined to gain insight into the relationship between aberrant gap junctional communication and spermatogenic impairment in the neoplastic testis. In uninvolved tubules with normal spermatogenesis, cx43 immunostaining was localized to the Sertoli-Sertoli junctional complex and cx26 was absent. In contrast, infiltrated tubules with spermatogonial arrest or CIS-only were negative for cx43, but displayed strong intracytoplasmic Sertoli cell staining for cx26. The Sertoli cells in these tubules re-expressed cytokeratin 18 (ck18), signifying a reversion to a less differentiated state. Western blot analysis for cx43 revealed a single immunoreactive band at 43 kD (normal spermatogenesis) and three bands at 43, 41, and 39 kD (impaired spermatogenesis with CIS or seminoma). For cx26, a doublet band at 26/28 kD (normal spermatogenesis) and an additional doublet band at 52/54 kD (impaired spermatogenesis with CIS or seminoma) were observed. The altered expression of cx26 and cx43 in Sertoli cells in testes infiltrated with CIS or seminoma suggests that a derangement in intercellular communication between Sertoli cells and between Sertoli cells and germ cells may play a role in the resulting spermatogenic impairment and possibly in the proliferation and neoplastic progression of CIS cells.     

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.     

The sertoli cell junctional complex: Structure and permeability to filipin in the neonatal and adult guinea pig

The development and maintenance of the Sertoli cell junctional complex were investigated in prepubertal and adult guinea pigs. To correlate the structure of the blood-testis barrier with its permeability, the polyene antibiotic filipin (a cholesterol-binding agent of low molecular weight: 570.70) was added to the fixative as a tracer visible in freeze-fracture replicas. Discontinuous zonules, intermediate junctions (i.e., adhering fasciae) and gap junctions all proved permeable to filipin in the two age groups. Only the continuous occluding zonules characteristic of the adult guinea pig's testis were impermeable to the tracer. In pubertal animals, the establishment of the blood-testis barrier coincided with the completion of the junctional strands in occluding zonules. The formation of occluding zonules was similar in the newborn and the adult. In the adult, the Sertoli cell junctional complexes contained three types of cell junctions: occluding, adhering, and gap junctions. The sequence of occluding and adhering junctions from the base to the apex of the epithelium was the reverse of that demonstrated in most epithelia. The impermeable continuous occluding zonules at the base showed parallel patterns of uninterrupted junctional strands, whereas the permeable discontinuous zonules found higher in the epithelium showed a meandering pattern of broken strands. Our observations indicate that (1) Sertoli cell junctional complexes form near the young germinal cells at the base of the seminiferous epithelium and break down near the older germinal cells toward the apex; (2) the various patterns and orientations of the junctional strands reflect, respectively, the different stages of disintegration of the occluding zonules and the conformation of the mature Sertoli cell to the irregular contours of the germinal cells; (3) there is no relationship between permeability and junctional strand orientation; and (4) the cellular contacts between Sertoli cells and germinal cells situated below the blood-testis barrier may represent the early stages of formation of junctional elements which ultimately become incorporated into the Sertoli cell junctional complex.     

Stability of the intra-epithelial component of the blood-testis barrier in epinephrine-induced testicular degeneration in Syrian hamsters

Adult male Syrian hamsters were given daily intraperitoneal injections of epinephrine (1.0 mg/kg) and papaverine, a vasodilator, (60 mg/kg) for a period of ten days. After the treatment period, lanthanum and horseradish peroxidase tracer studies were used to examine the intra-epithelial component of the blood-testis barrier. Degenerating tubules often exhibited only Sertoli cells and spermatogonia, or Sertoli cells alone. Sertoli cell processes in the degenerating tubules often arched out from the main cell body to make contact with other Sertoli cell processes, forming a series of vacuole-like spaces in the germinal epithelium, adluminal to the Sertoli-Sertoli junctions. At the site of contact between these arching Sertoli cell processes one to eight tight junctions had formed, with hexagonal arrays of Sertoli cell cytoplasmic filaments located immediately adjacent to these junctions. Cisternae of the Sertoli cell endoplasmic reticulum lay deep to the layer of cytoplasmic filaments. It appeared that these junctions had originated after the expulsion of the germinal elements of the seminiferous epithelium. Penetration of the tracers in the degenerating seminiferous tubules was prevented by what appeared to be normal Sertoli-Sertoli junctions located between apposed Sertoli cells, adluminal to the remaining spermatogonia when these resisted degeneration, or just adluminal to the basal lamina in those tubules in which spermatogonia were absent.     

Immunofluorescence localization of vinculin in ectoplasmic (“junctional”) specializations of rat sertoli cells

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

Observations on the inter-relationships of Sertoli cells at the level of the blood-testis barrier: Evidence for formation and resorption of Sertoli-Sertoli tubulobulbar complexes during the spermatogenic cycle of the rat

Structures termed tubulobulbar complexes are known to be formed by adjoining Sertoli cells at the level of the blood-testis barrier (Russell and Clermont, 1976). Here, long (2–4 μm) tubular evaginations of one Sertoli cell, which end in bulbous dilations, are seen in corresponding invaginations of a neighboring Sertoli cell. In most regions of the tubular and bulbous portions of the complex, the Sertoli plasma membranes were found to be separated by a 4–5-nm intercellular space, but in some areas the membranes converged to form tight and gap junctions. The numbers, distribution and properties of tubulobulbar complexes were studied in relation to the cycle of the seminiferous epithelium. From the data obtained it was concluded that tubulobulbar complexes develop and undergo regressive changes during the spermatogenic cycle. Most complexes arise during the early stages of the cycle (Stages II-V) and develop large bulbous endings. Developing tubulobulbar complexes consist of short evaginations of one Sertoli cell which face a bristle-coated pit of the opposing Sertoli cell. At midcycle (Stages VI-VII) most show regressive changes and are eventually resorbed as a consequence of the action of nearby Sertoli lysosomes. Once resorbed, the probability of seeing a tubulobulbar complex in thin sections decreases from 4- to 8-fold. The few tubulobulbar complexes which remain past this period (Stages VII-XIV-I) usually lack bulbous endings and are frequently seen above type A spermatogonia. The data suggest that small fragments of cytoplasm and plasma membrane (including junctional surfaces) are lost from one Sertoli cell as a result of the degradative processes occurring in a neighboring Sertoli cell. Tubulobulbar resorption is discussed in relation to the impending breakdown of the blood-testis barrier above spermatocytes as these cells move upward. The possible significance of the cyclic resorption of tight and gap junctional sites between Sertoli cells is also discussed.     

Anchoring device between Sertoli cells and late spermatids in rat seminiferous tubules

Near the end of spermiogenesis, the late spermatids remain attached to the superficial layer of the seminiferous epithelium for an appreciable period of time (i.e., 3 to 4 days). The sickle-shaped heads of the spermatids are embedded in an apical process of Sertoli cell cytoplasm which is connected to the rest of the cell by a narrow stalk. In the concavity of the head several long (2–3 μm) and very narrow (50 nm) tubular projections of the spermatid's plasma membrane invaginate the Sertoli cell cytoplasm. These tubular processes terminate by a bulbous swelling which may measure up to 1 μm in diameter. Along the process the plasma membrane of the Sertoli cell is closely apposed to the spermatid's membrane, the intercellular space being only 6–8 nm wide. In the Sertoli cytoplasm immediately surrounding the tubular portion of the structure there is an accumulation of filamentous material, while next to the bulbous extremity there are, at a short distance, smooth surfaced cisternae of endoplasmic reticulum. The whole structure was referred to as a tubulobulbar complex. These complexes, of which there are up to 24 per spermatid, appear as these cells complete their migration toward the apex of the Sertoli cells. They disappear just before the release of the spermatids in the lumen of the seminiferous tubule as a result of the fragmentation of the spermatid's plasma membrane followed by a resorption of the Sertoli plasma membrane. Morphological evidence suggests that the tubulobulbar complexes serve as anchoring devices that retain the spermatids at the surface of the seminiferous epithelium while their dissolution contributes in part to the process of spermiation. Similar tubulobulbar complexes were also formed by the plasma membranes of two adjacent Sertoli cells close to the Sertoli-Sertoli tight junctions near the tubular limiting membrane.     

Three-dimensional reconstruction of a rat stage V Sertoli cell: III. A study of specific cellular relationships

Specific Sertoli–Sertoli and Sertoli–germ-cell contacts and/or junctions were investigated employing micrographs used to reconstruct serially a model of a rat stage V Sertoli cell. The Sertoli–Sertoli junctional contact areas occurred in a belt-like arrangement near the base of the Sertoli cell. This configuration is consistent with their proposed function as a sealing element limiting the passage of materials toward the tubular lumen. Sertoli ectoplasmic specializations also formed a continuous belt, or band, around the reconstructed cell at the junctional contact area. Eighteen Sertoli–Sertoli tubulobulbar complexes were found; some (12 in number) invaginated the reconstructed cell, while others (6) emanated from it. Of 37 round germ cells that were sectioned in their entirety and adjoined the reconstructed cell, 23 displayed desmosome–gap junctions with either the reconstructed cell or an adjoining cell. Since there were multiple junctions connecting some germ cells to Sertoli cells, the total number of junctions was much greater (35). Desmosome–gap junctions of the Sertoli cell were numerous connecting pachytene spermatocytes, less numerous connecting type B spermatogonia, and even less numerous connecting step 5 spermatids; and none was seen joining Sertoli cells with elongate spermatids. Most desmosome-gap junctions join germ cells to the body of the Sertoli cell at its basal aspect. Their numbers and position indicate that they play a role in the maintenance of the integrity of the seminiferous epithelium and may provide a route for cell-to-cell communication. Ectoplasmic specializations of the reconstructed cell were seen facing only 3 of 37 round germ cells, and 7 ectoplasmic specializations from adjoining Sertoli cells faced these germ cells, all of which were step 5 spermatids. That there were no ectoplasmic specializations facing pachytene cells indicates that ectoplasmic specializations are not acquired as these cells pass through Sertoli–Sertoli junctions, but are acquired later in spermatogenesis.