Deep-etch visualization of the Sertoli cell (blood-testis) barrier in the boar.

The Sertoli cell (blood-testis) barrier in the boar was visualized by the freeze-fracture, deep-etch, rotary-replication technique. Three kinds of cross-bridging structures were clearly recognized in the following three ectoplasmic specialization (ES) regions; (1) cross-bridges in the intercellular space between adjacent Sertoli cell membranes; (2) cross-bridges in the space between the Sertoli cell membrane and microfilament bundles; and (3) cross-bridges in the space between microfilament bundles and subsurface cisternae. Results from immunolocalization, vinculin and alpha-actinin were recognized in the Sertoli cell barrier. Our findings show that these structural elements of the Sertoli cell barrier are held together by these cross-bridging structures, and provide important morphological evidence that implicates the ES in the dynamic function of the microfilament bundles of the Sertoli cell barrier.     

金鱼精巢支持细胞间连接和血睾屏障

Freeze-fracture and etching technique combined with thin sectioning and lanthanum impregnation has been used for the study of Sertoli cell junctions and the blood-testis barrier formation in goldfish testis with lobular organization. Some observations and results are first given in this paper. The results of experiments can be summarized as the following: 1). Sertoli cell junctions are compound junctions of tight junctions, desmosomes and gap junctions. Tight junctions usually appear as parallel or network like ridges on the P face and fine grooves on the E face at the freeze-etching replicas. Desmosomes and gap junctions often are located between or nearby the ridges of tight junctions. In addition, endoplasmic reticulum cristae near the junction area can also be observed. 2). The number, area and density of each individual junction vary with the development and differentiation stages of germinal cells in the cyst. 3). Tight junctions can be observed at any stage during germinal cell differentiation through the period of spermatogenesis and spermiogenesis. However, they appear morphologically different as type I and type II. 4). Lanthanum can partially penetrate into the intercellular spaces of spermatogonium and early primary spermatocyte but can't penetrate after the stage of late primary spermatocyte. 5). The blood-testis barrier formation starts at the stage of pachytene spermatocytes. The formation of the blood-testis barrier is the result of the development of the tight junction from type I to type II.     

Effects of vasectomy on the blood-testis barrier of the hamster.

The effects of vasectomy on the blood-testis and blood-epididymal barriers to 3H2O, [3H]inulin, and [14C]urea were examined by study of the radioactivity appearing in micropuncture samples of fluids from the seminiferous tubules and cauda epididymidis. By 4 months after vasectomy, there were changes in the blood-seminiferous tubule barrier to [3H]water and [14C]urea (increased entry) and in the blood-epididymal barrier to [3H]water and [3H]inulin (increased entry) and to [14C]urea (decreased entry). These subtle changes could have an impact on spermatogenesis and/or sperm maturation after vasectomy.     

The blood-testis barrier in Aphanius dispar (Teleostei)

Summary Thin sections of normal testes from the cyprinodont Aphanius dispar were studied by electron microscopy after intravascular injection of live specimens with horseradish peroxidase. The intercellular space in the spermatogenic cysts is marked differently by the tracer according to the degree of differentiation of the germ cells. Spermatogonia and gonocytes undergoing meiosis are surrounded by a dark band of the marker. This band gradually disappears during spermiogenesis. In cysts containing ripe spermatozoa, the marker penetrates a short distance between the bases of adjoining Sertoli cells bordering the cysts, but is arrested by tight junctional complexes near the lumina of the cysts. The tight junctions between the Sertoli cells provide a permeability barrier between the vascular spaces of the stroma and the lumina of ripe cysts.Abbreviations BM basement membrane - BTB blood-testis barrier - HRP horseradish peroxidase This research was supported by a grant from the National Council for Research and Development, Israel, and the GKSS Geesthacht-Tesperhude, Federal Republic of Germany     

Formation of the blood-testis barrier in the rabbit

Summary The time of establishment of the blood-testis barrier in the rabbit was studied by electron microscopy using lanthanum nitrate. This electron-dense tracer was present in the intercellular spaces in all regions of the seminiferous cords in 7 to 9-week-old animals. In 10 and 11-week-old rabbits, the penetration of lanthanum nitrate was restricted to the basal region of the seminiferous cords. Closer examination revealed the presence of numerous tight junctions between adjacent Sertoli cells. The morphological appearance of these junctions was similar to those described previously in other mammals. Entry of the tracer substance was restricted at these junctions. Pachytene germ cells, which reside beyond the junctions, were never surrounded by the tracer. Based on our observations it was concluded that the blood-testis barrier in the rabbit is formed between the 9th and 10th postnatal week, and that it is functionally effective by the 10th week.     

Studies on the permeability of the blood-testis barrier in stainless steel-administered mice

Previous trials performed in our laboratory have demonstrated that stainless steel corrosion products induced morphological changes in mice seminiferous cells. In this study the functional nature of the blood-testis barrier was investigated, in view of the extesive use of this metallic alloy in orthopaedic surgery. The uptake of horseradish peroxidase by the seminiferous tubules was used as a tracer method being exposed in vitro for different periods of time and observed at the ultrastructural level. Both boundary cells as well as Sertoli cells of experimental group absorbed the tracer, as manifested by vesicles and vacuoles filled with the reaction product, evidencing its role within the testis. Germ cells were also able in the uptake of this macromolecule. However, the tracer did not spread into the junctional complexes between neighboring Sertoli cells, at any interval of time used in this study, demonstrating the functional integrity of this barrier.     

Effects of cytochalasin D on fusion of cells by HVJ (Sendai virus).

Fusion of cells mediated by HVJ was inhibited completely with 5 μg/ml or more of cytochalasin D (CD). With cytochalasin, HVJ-cell interaction at 0 °C proceeded as well as without cytochalasin; HVJ was adsorbed to cell surfaces and the cells agglutinated together. Then the virus particles were enfolded with cell membranes, which resulted in the disappearance of hemadsorption activity on the cell surfaces. When the cell-virus complex was incubated at 37 °C, the early reactions proceeded as well as without cytochalasin; the hemadsorption activity reappeared on the cell surfaces, the viral envelopes fused with cell membranes at the same degree as without cytochalasin, and a stage sensitive to sodium azide appeared as in a control without cytochalasin. But cell-to-cell fusion did not occur in the presence of cytochalasin; cells were dissociated freely from the cell aggregates during incubation. This indicates that cell-to-cell fusion was inhibited but HVJ envelope to cell membrane interactions proceeded well on incubation at 37 °C. These findings suggest that viral envelope-cell membrane fusion and cell-cell fusion are separable, and participation of a cytoskeleton system including microfilaments in the cells is essential for cell-cell fusion.     

Observations on the blood-testis barrier in a frog and a salamander

Cell and Tissue Research - A blood-testis barrier has been demonstrated in a frog, Rana esculenta, and in a salamander, Salamandra salamandra, using lanthanum as an electron-dense marker during...     

Disruption of blood-testis barrier dynamics in ether-lipid-deficient mice

One of the major roles of Sertoli cells is to establish the blood-testis (Sertoli cell) barrier (BTB), which is permanently assembled and disassembled to accommodate the translocation of leptotene spermatocytes from the basal into the adluminal compartment of the seminiferous epithelium and to guarantee completion of meiosis and spermiogenesis. Recently, we have demonstrated spermatogenesis to be arrested before spermatid elongation in Gnpat-null mice with selective deficiency of ether lipids (ELs) whose functions are poorly understood. In this study, we have focused on the spatio-temporal expression of several BTB tight-junctional proteins in the first wave of spermatogenesis to obtain insights into the physiological role of ELs during BTB establishment and dynamics. Our data confirm the transient existence of Russell’s intermediate or translocation compartment delineated by two separate claudin-3-positive luminal and basal tight junctions and reveal that EL deficiency blocks BTB remodeling. This block is associated with (1) downregulation and mistargeting of claudin-3 and (2) impaired BTB disassembly resulting in deficient sealing of the intermediate compartment as shown by increased BTB permeability to biotin. These results suggest that ELs are essential for cyclic BTB dynamics ensuring the sluice mechanism for leptotene translocation into the adluminal compartment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by the German Research Foundation (grants Go 432/2-1, Ju 166/3-1, and Sa 172/1-1).     

Transport of Poly(n-butylcyano-acrylate) nanoparticles across the blood-brain barrier in vitro and their influence on barrier integrity

In previous studies it was shown that polysorbate 80(PS80)-coated poly(n-butylcyano-acrylate) nanoparticles (PBCA-NP) are able to cross the blood–brain barrier (BBB) in vitro and in vivo. In order to explore and extend the potential applications of PBCA-NP as drug carriers, it is important to ascertain their effect on the BBB. The objective of the present study was to determine the effect of PS80-coated PBCA-NP on the BBB integrity of a porcine in vitro model. This has been investigated by monitoring the development of the transendothelial electrical resistance (TEER) after the addition of PBCA-NP employing impedance spectroscopy. Additionally, the integrity of the BBB in vitro was verified by measuring the passage of the reference substances ¹⁴C-sucrose and FITC-BSA after addition of PBCA-NP. In this study we will show that the application of PS80-coated PBCA-NP leads to a reversible disruption of the barrier after 4 h. The observed disruption of the barrier could also be confirmed by ¹⁴C-sucrose and FITC-BSA permeability studies. Comparing the TEER and permeability studies the lowest resistances and maximal values for permeabilities were both observed after 4 h. These results indicate that PS80-coated PBCA-NP might be suitable for the use as drug carriers. The reversible disruption also offers the possibility to use these particles as specific opener of the BBB. Instead of incorporating the therapeutic agents into the NP, the drugs may cross the BBB after being applied simultaneously with the PBCA-NP.     

Interleukin 1 alpha (IL1A) is a novel regulator of the blood-testis barrier in the rat

Throughout spermatogenesis, leptotene spermatocytes must traverse the blood-testis barrier (BTB) at stages VIII-XI to gain entry into the adluminal compartment for continued development. However, the mechanism underlying BTB restructuring remains somewhat elusive. In this study, interleukin 1 alpha (IL1A) was administered intratesticularly to adult rats in order to assess its effects on spermatogenesis. IL1A was shown to perturb Sertoli-germ cell adhesion, resulting in germ cell loss from approximately 50% of seminiferous tubules by 15 days posttreatment. Equally important, the functional integrity of the BTB was compromised when inulin-fluorescein isothiocyanate was detected in the adluminal compartment of the seminiferous epithelium following its administration via the jugular vein. Interestingly, IL1A did not affect the steady-state levels of proteins that confer BTB function, namely OCLN, CLDN1, F11R, TJP1, and CDH2. Instead, the localizations of OCLN, F11R, and TJP1 in the seminiferous epithelium were altered; these proteins appeared to move away from sites of cell-cell contact. Moreover, IL1A was shown to perturb the orderly arrangement of filamentous actin at the BTB and apical ectoplasmic specialization with distinct areas illustrating loss of actin filaments. Taken collectively, these results suggest that IL1A-induced BTB disruption is not mediated via the reduction of target protein levels. Instead, IL1A's primary cellular target appears to be the Sertoli cell actin cytoskeleton. It is possible that localized production of IL1A by Sertoli and/or germ cells in vivo results in BTB restructuring, and this may facilitate the movement of leptotene spermatocytes across the BTB.     

Changes in the blood-testis barrier as a result of streptococcal sensitization

Permeability of the histo-hematic barrier in the guinea pig and mouse testes immunized with streptococci has been studied radiometrically and luminescent microscopically with application of labelled bovine serum albumin, human gamma-globulin and blue dye preparations. The permeability of the hemato-testicular barrier increases on the 7th--30th day after immunization. Test-preparations penetrate from the vessels of the intercanalicular connective tissue layers through the membrane of the convoluted tubules. The testicular autoantigen is revealed in blood. The disturbance in permeability of the hemato-testicular barrier precedes or coincides in time with the developing destruction of the testicular spermatogenic elements.     

Effect of efferent duct ligation on the function of the blood-testis barrier in rats

The function of the blood-testis barrier has been assessed from the ratio of the Cr-EDTA space in the parenchyma to the measured interstitial volume in the testes of rats at various times after unilateral ligation of the efferent ducts. The barrier remained effective during the phase of fluid accumulation and testicular mass gain, which was linear for at least 24 h, but the testis mass began to decrease between 32 and 40 h after efferent duct ligation, and the Cr-EDTA space at 40 and 48 h after efferent duct ligation exceeded the volume of the interstitial tissue. This finding indicated that, at these times, the barrier to Cr-EDTA, which is normally excluded from the tubules, had broken down and the marker was entering the tubules. Thereafter, the Cr-EDTA space decreased again to be less than the interstitial tissue volume, indicating a restoration of the barrier function, although degeneration of the seminiferous epithelium continued to become more obvious. The present study is the first report of a reversible breakdown of the barrier, but the relevance of the breakdown to the effects on spermatogenesis requires further study.     

Observations on the effects of cytochalasin B and cytochalasin D on ADP- and chymotrypsin-treated platelets

Cytochalasin B has been reported to inhibit fibrinogen binding and aggregation of rabbit platelets in response to ADP. The present study was designed to ascertain whether cytochalasins B and D inhibit aggregation by interfering with the exposure of fibrinogen receptors or more directly by inhibiting binding to available receptors. Aspirin-treated, washed, human platelets stimulated with ADP or chymotrypsin were used for these studies. Neither cytochalasin B nor D significantly inhibited the binding of fibrinogen to chymotrypsin-treated platelets when these agents were added to platelet suspensions before (16 +/- 8% (mean +/- SD) inhibition, N = 8), or after (15 +/- 10% inhibition, N = 13) chymotrypsin treatment, i.e., before or after fibrinogen receptor exposure. This apparent lack of cytoskeletal involvement was consistent with the observation that chymotrypsin-treated platelets were unable to retract reptilase-induced fibrin clots, an activity that was restored by adding ADP. In contrast, incubating platelets with either cytochalasin B or D for 30 min before or after stimulation with ADP decreased fibrinogen binding by 42 +/- 16% (N = 13) and 27 +/- 11% (N = 8), respectively, compared to DMSO-treated controls. Platelets stimulated with ADP and incubated with DMSO for 30 min, however, became refractory and aggregated poorly in response to a second dose of ADP. In comparison, platelets stimulated with ADP, but incubated with cytochalasin B or D, aggregated more extensively when stimulated by a second dose of ADP despite diminished fibrinogen binding. The data suggest (1) microfilament polymerization is important not only for the exposure of fibrinogen receptors by ADP, but also for preserving the ability of exposed receptors to bind fibrinogen, (2) exposure of fibrinogen receptors by chymotrypsin is not accompanied by significant cytoskeletal activation, and (3) cytochalasins may impart partial protective effects against the development of ADP-induced refractoriness.     

Regulation of blood-testis barrier dynamics by focal adhesion kinase (FAK): An unexpected turn of events

The blood-testis barrier (BTB) is conferred by co-existing tight junctions (TJs), basal ectoplasmic specialization (basal ES), desmosome-like junctions and gap junctions (GJs) between adjacent Sertoli cells near the basement membrane in the seminiferous epithelium. While the concept of the BTB has been known for more than a century and its significance to spermatogenesis discerned for more than five decades, its regulation has remained largely unknown. Recent studies, however, have demonstrated that focal adhesion kinase (FAK), a modulator of the integrin-based signaling that plays a crucial role on cell movement, apoptosis, cell survival and gene expression at the focal adhesion complex (FAC, also known as focal contact, a cell-matrix anchoring junction type), is an integrated component of the BTB, associated with the TJ-integral membrane protein occludin and its adaptor zonula occludens-1 (ZO-1). Herein, we summarize recent findings in the field regarding the significance of FAK in conferring BTB integrity based on some unexpected observations. We also critically discuss the role of FAK in regulating the timely "opening" and "closing" of the BTB to facilitate the transit of primary preleptotene spermatocytes across the BTB at stage VIII of the seminiferous epithelial cycle of spermatogenesis. Lastly, we propose a working model, which can be used to design future functional experiments to explore the involvement of FAK in BTB dynamics by investigators in the field.