Cell junction dynamics in the testis: Sertoli-germ cell interactions and male contraceptive development

Spermatogenesis is an intriguing but complicated biological process. However, many studies since the 1960s have focused either on the hormonal events of the hypothalamus-pituitary-testicular axis or morphological events that take place in the seminiferous epithelium. Recent advances in biochemistry, cell biology, and molecular biology have shifted attention to understanding some of the key events that regulate spermatogenesis, such as germ cell apoptosis, cell cycle regulation, Sertoli-germ cell communication, and junction dynamics. In this review, we discuss the physiology and biology of junction dynamics in the testis, in particular how these events affect interactions of Sertoli and germ cells in the seminiferous epithelium behind the blood-testis barrier. We also discuss how these events regulate the opening and closing of the blood-testis barrier to permit the timely passage of preleptotene and leptotene spermatocytes across the blood-testis barrier. This is physiologically important since developing germ cells must translocate across the blood-testis barrier as well as traverse the seminiferous epithelium during their development. We also discuss several available in vitro and in vivo models that can be used to study Sertoli-germ cell anchoring junctions and Sertoli-Sertoli tight junctions. An in-depth survey in this subject has also identified several potential targets to be tackled to perturb spermatogenesis, which will likely lead to the development of novel male contraceptives.     

Unique features of the cauda epididymidal epithelium of hibernating bats may promote sperm longevity

Measurements of extremely high osmolalities in cauda epididymidal fluids of hibernating bat species led to an investigation of the junctional complex morphology of the epithelium of this sperm storage site. Freeze fracture replicas revealed the presence, at certain times of the year, of a tight junction architecture that resembled that traditionally thought to be exclusive to the blood-testis barrier, the strongest permeability barrier in the body. It is hypothesized that seasonal establishment of these specialized Sertoli cell-like tight junctions is necessary to the maintenance of the high osmotic state of the luminal environment, allowing for the prevention of dilution of its contents by paracellular routes and its protection from bursting under the osmotic pressure contained within. © 1993 Wiley-Liss, Inc.     

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.     

A novel perspective: The occluding zonule encircles the apex of the sertoli cell as observed in birds

The modulation of Sertoli cell junctions was studied in the non-seasonal rooster (Gallus domesticus) and in the seasonally breeding mallard duck (Anas platyrynchos anatidae) using thin sectioning, a junction permeability tracer, and freeze-fracture replication. During the active spermatogenic phase, the junctions of the duck appeared similar to those of the rooster, therby establishing the duck as an avian model of seasonal modulation of Sertoli cell junctions. As with mammalian seasonal breeders, during the active phase, occluding, gap, and adhering junctions formed a junctional complex all along the long axis of the Sertoli cell. Unlike in mammals, however, no 7-nm filaments were associated with the occluding junctions. An occluding zonule encircled the Sertoli cell apico-lateral membrane domain situated above the young germ cells, and constituted a barrier to the entry of lanthanum in the basal third of the seminiferous epithelium. Toward the basal side, forming focal junctions were located on the lateral Sertoli cell membrane domain facing the young germ cells. Toward the apical side, dismantling focal junctions were located on the apical Sertoli cell membrane domain facing the older germ cells. During the duck's testicular regression, 7-nm filaments were associated with an occluding junction. In freeze-fracture replicas, each junction was formed by a continuous junctional strand that encircled the apex of the cell. The strands composed a delicate narrow meshwork: an occluding zonule. The blood-testis barrier was localized near the apex of the epithelium. The seasonal reduction in the number of the strands and the changes in their orientation did not coincide with a change in the permeability of the occluding zonule to lanthanum. In addition, the cyclic disappearance of junction-associated filaments was not correlated with a change in the permeability of the junctions but with a change in the affinity of junctional particles for one or the other fracture face. It is proposed that the Sertoli cell plasma membrane domains situated apical and basal with respect to the occluding zonule be considered apical and lateral, respectively. The remaining domain facing the basement membrane would therefore be called basal. In the duck, the occluding zonule is not seasonally shifted from the base to the apex of the Sertoli cell. Instead, it remains stationed above the younger germ cells throughout the year. The illusory shift in the location of the blood-testis barrier during the active phase actually results from the formidable expansion of the Sertoli cell apical membrane domain above the occluding zonule to accommodate the development of germ cells. It follows that the occluding zonules forming the blood-testis barrier in the nonseasonal breeders actually encircle the apices of the Sertoli cells although they occur in the basal third of the seminiferous epithelium.     

Junctional complexes of the blood-testis barrier in the Japanese quail (Coturnix Coturnix japonica)

This study investigated the developmental changes in the adherens junctions, gap junctions, as well as tight junctions forming the blood-testis barrier (BTB) in Japanese quail (Coturnix Coturnix japonica) testis. Testicular tissue from pre-pubertal, pubertal, adult, and aged Japanese quail were examined by immunohistochemistry and transmission electron microscopy (TEM). The tight junction proteins claudin-3, claudin-11, occludin and zonula occludens-1 (ZO-1), were generally localised in the cytoplasm of Sertoli cells, spermatogonia, and spermatocytes of pre-pubertal, pubertal, some adult birds. The adherens junction protein E-cadherin had a similar distribution pattern. During pre-pubertal development, the gap junction protein connexin-43 (Cx43) was only localised between Leydig cells in the testicular interstitium. However, TEM revealed the presence of gap junctions between cells of the seminiferous epithelium as early as the pre-pubertal stage. Furthermore, TEM confirmed the presence of tight and adherens junctions in the seminiferous epithelia of all age groups. The findings of this study document age-related differences in the immunolocalisation and intensity of the junctional proteins and the ultrastructure of the junctional complexes forming the BTB in quail testes. Additionally, the junctional complexes forming the BTB in the Japanese quail are well established prior to puberty. This study provides baseline information for the future evaluation of pathological changes in the BTB of avian species at different developmental stages.     

The blood-testis barrier and its formation relative to spermatocyte maturation in the adult rat: a lanthanum tracer study

An electron-opaque substance, lanthanum, was utilized to determine when germ cells of the rat first cross the blood-testis barrier in adult spermatogenesis. Intravascularly perfused lanthanum was shown to surround all spermatogonia. Intravascularly perfused lanthanum was shown to surround all spermatogonia, preleptotene spermatocytes and early leptotene spermatocytes of Stage IX in the adult rat testis. Lanthanum was excluded from the spaces around more mature cells by newly-formed tight junctions between adjoining Sertoli processes. These processes had previously intervened between the leptotene cells and the basal lamina. The results are in close agreement with those of a previous study (Russell, '77a) which indicated that leptotene cells are the first cells of adult spermatogenesis to enter the intermediate compartment and to reside beyond a permeability barrier.     

慢性镉中毒小鼠生精上皮的超微结构观察和立体定量分析

本用透射电镜观察并结合立体定量法研究了慢性镉中毒时小鼠睾丸生精上皮的超微结构。结果为：（1）支持细胞明显受损，细胞核增大，细胞器出现变性变化，有的支持细胞呈现坏死现象，（2）各级生精细胞有不同程度损伤，生精小管管腔内常见未成熟精子和畸形精子。（3）支持细胞的细胞连接被破坏，血－生精小管屏障部分瓦解，本还对慢性镉中毒时影响精子发生的机理进行了初步探讨，认为支持及其细胞连接的结构和功能异常可能是生精障碍的主要原因。     

Cyclic modulation of sertoli cell junctional complexes in a seasonal breeder: The mink (Mustela vison)

The development and modulation of Sertoli cell junctions was studied in newborn and adult mink during the active and inactive spermatogenic phases. The techniques used were electron microscopy of freeze-fractured replicas and thin sections of tissues infused with horseradish peroxidase as a junction permeability tracer. In the newborn, freeze-fractured developing junctions had either spherical or fibrillar particles. In addition, Junctional domains where particles were associated preferentially with the E-face, and others where particles were associated preferentially with the P-face, were found developing either singly or conjointly within a given membrane segment, thus yielding a heterogeneous Junctional segment. Coincidently with the development of a tubular lumen and the establishment of a competent blood-testis barrier, Junctional strands were composed primarily of particulate elements associated preferentially with the E-face. In adult mink during active spermatogenesis, cell junctions were found on the entire lateral Sertoli cell plasma membrane from the basal to the luminal pole of the cell. In the basal third of the Sertoli cell, membranous segments that faced a spermatogonium or a migrating spermatocyte displayed forming tight, gap, and adherens junctions. In the middle third, abutting membrane segments localized above germ cells were involved in continuous zonules and in adherens junctions. In the apical or luminal third, the zonules were discontinuous, and the association of Junctional particles with the E-face furrow was lost. Gap junctions increased in both size and numbers. Junctional vesicles that appeared as annular gap and tight-junction profiles in thin sections or as hemispheres in freeze-fracture replicas were present. Reflexive tight and gap junctions were formed through the interaction of plasma membrane segments of the same Sertoli cell. Internalized Junctional vesicles were also present in mature spermatids. During the inactive spermatogenic phase, cell junctions were localized principally in the basal third of the Sertoli cell; Junctional strands resembled those of the newborn mink. During the active spermatogenic phase, continuous zonules were competent in blocking passage of the protein tracer. During the inactive phase the blood-testis barrier was incompetent in blocking entry of the tracer into the seminiferous epithelium. It is proposed that modulation of the Sertoli cell zonules being formed at the base and dismantled at the apex of the seminiferous epithelium follows the direction of germ cell migration and opposes the apicobasal direction of junction formation reported for most epithelia. The arrest of spermatogenesis coincides with dramatic changes in the dynamic modifications of Sertoli cell zonules.     

Effects of cytochalasin D on the integrity of the Sertoli cell (blood-testis) barrier

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150–500 μM CD; 0.5–12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture experiments, significantly more radiolabelled inulin entered the lumen of seminiferous tubules from CD-treated animals than from those exposed to vehicle. Collectively, the data suggest that CD treatment alters the functional integrity of the Sertoli cell barrier and that this may be the result of disruption of microfilaments associated with the barrier.     

Barrier function of microvessels and roles of glial cell line-derived neurotrophic factor in the rat testis

The expression and localization of glial cell line-derived neurotrophic factor (GDNF) and its receptor GFR alpha1 in the testis were examined, because the blood-testis barrier is a well-known tissue barrier and we previously reported that GDNF reduced the endothelial permeability of the blood-brain barrier (BBB). Five minutes after intravenous injection of Evans blue (molecular weight, 960.6) or fluorescent dextran (molecular weight 10000 and 70000), Evans blue was observed outside microvessels of the testis, whereas the fluorescent dextran was not. Immunohistochemically, GDNF was detected in alpha-smooth muscle actin-positive cells around the seminiferous tubules and in microvessels. On the other hand, GFR alpha1 was detected in endothelial cells in the interstitial space, as well as in spermatocytes. Although occludin was positive in Sertoli cells and endothelium, claudin-5 was localized only in the endothelium of the microvessels. Thus, it became very clear that the microvessels in the testis possessed relatively impermeable tight junctions, and that the alpha-smooth muscle actin-positive cells secreted GDNF, which receptor was expressed in endothelial cells. Because this relation between GDNF and GFR alpha1 is similar to that observed in the BBB, we hypothesize that GDNF is a general regulator of tight junctions of the endothelium forming a blood-tissue barrier in a paracrine fashion.     

Movement of spermatocytes from the basal to the adluminal compartment of the rat testis

The progressive movement of primary spermatocytes from the basal to the adluminal compartment of the seminiferous tubule was studies after testes were fixed with standard and hypertonic solutions. In stages VI, VII and VIII of the cycle (classification of Leblond and Clermont, '52), preleptotene spermatocytes were observed within the basal compartment of the seminiferous tubule. Resting on the basal lamina, these cells were bound tightly to neighboring Sertoli cells by desmosome-like junctions. In late stage VIII and early stage IX, basal processes of Sertoli cells were observed between the newly formed leptotene cells and the basal lamina, and in stage IX, the Sertoli processes met to form a junction of the zonula adherens type. This junction formed a permeability barrier which restricted the free access of fixative into the spaces around leptotene cells. Evidence for this was found in the absence of the shrinkage artifact produced with hypertonic solutions in earlier stages. In longitudinal sections, the permeability barrier was first observed in an area of the tubule in which sperm release was also taking place. In mid- stage IX and in stage X, Sertoli-Sertoli junctional specializations formed de novo below the leptotene spermatocyte, while those from the preceding stages, present above the leptotene spermatocytes, remained intact. Thus, tight junctions were in evidence for a considerable period of the time, both above and below the leptotene spermatocytes. At no time in the process of germ cell movement toward the lumen did these cells exhibit evidence of amoeboid movement or lose desmosome-like contacts with the surrounding Sertoli cells. From this study it is concluded that the Sertoli cells play an active role in the transfer of spermatocytes to the adluminal compartment. A transient intermediate compartment of the seminiferous tubule is described, one which allows for the continual maintenance of the blood-testis barrier during transit of spermatocytes from the basal to the adluminal compartment.     

The postnatal development of the junctional complexes of the mouse Sertoli cells as revealed by freeze-fracture

Mouse testes of newborn to adult were examined by freezefracture. Between the newborn Sertoli cells, gap junctions consisting of aggregations of the intramembranous particles (about 8 nm in diameter) are frequently found. Some of the junctions are about 1 μm in diameter and show particle-free regions in the aggregation. Linear arrangements of a few particles, which appear to be the initial formation of the occluding junctions, are seen in the newborn Sertoli cells. The occluding junctions are arranged in a meshwork, in which the gap junctions are situated between the stages of newborn to six days of age. The particles of the occluding junctions are predominantly located on the B face in the center of the groove instead of the A face of the ridge. The occluding junctions do not appear to surround the entire circumference of the Sertoli cell of the 6-day-old mouse. The gap junctions decrease in size. In later stages, many parallel occluding junctions (up to forty in number) are found over one Sertoli cell surface and are distributed circumferentially around the entire cell surface, indicating establishment of the blood-testis barrier. The occluding junctions dominate and the gap junctions diminish in number as development proceeds.     

Cyclic formation and decay of the blood-testis barrier in the mink (Mustela vison), a seasonal breeder

The correlations between the germ cell population and the blood-testis barrier were studied during puberty and throughout the reproductive cycle in a seasonal breeder, the mink. A classification of 12 stages, corresponding to the cellular associations appearing during the cycle of the seminiferous epithelium, was proposed and used to identify the stages of the cycle in pubertal mink. In adult mink, the reproductive cycle was divided into two spermatogenic phases–an active phase lasting 9 months, and an inactive phase lasting 3 months. The active spermatogenic phase was broken down into three distinct periods: the first spermatogenic wave, the peak of spermatogenic activity, and the last spermatogenic wave. Degenerating germ cells were found in comparable and relatively low proportions during puberty and during the first and last spermatogenic waves of the adult reproductive cycle. The permeability of the blood-testis barrier to intravascularly infused electron-opaque tracers (i.e., horseradish peroxidase and lanthanum) was tested at the time of the first spermatogenic wave at puberty and throughout the reproductive cycle of the adult. The relationship between epithelial permeability and germ cell populations prevailing during puberty and during the first and last spermatogenic waves of the adult active phase was the same. During puberty, the establishment of the blood-testis barrier did not coincide with the appearance of a particular step of meiosis but was correlated with the development of a tubular lumen. In adult mink, the barrier cyclically decayed during the last wave of the active spermatogenic phase and reformed during the first wave of the next active phase. The decay and the reformation of the barrier were not coincident with the appearance or disappearance of a particular generation of the germ cell population from the seminiferous epithelium but were correlated with cyclic cytological changes in Sertoli cells and the rhythmic development and occlusion of the lumen. During the peak months of the active spermatogenic phase, however, a blood-testis barrier secluded spermatogonia and young spermatocytes from older generations of germ cells. It is concluded that (1) during puberty and also during the first and last spermatogenic wave of the adult mink reproductive cycle, the development of germ cells is possible in the absence of a competent, impermeable blood-testis barrier, and (2) the transient presence of a permeable epithelial barrier does not initiate an autoimmune response of sufficient magnitude to cause destruction of the seminiferous epithelium.     

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.     

Effects of immunization with Freund's complete adjuvant and isologous spermatozoa on the seminiferous epithelium and blood-testis barrier in guinea pigs

In order to gain insight into the earliest pathological changes underlying the development of autoimmune aspermatogenic orchitis (AIAO) the blood-testis barrier was studied by light and electron microscopy, freeze-etching, and cytochemical techniques early (from 1 to 8 days after adjuvant treatment or isoimmunization). At later times (16 to 21 days) the study was carried out by light microscopy only. Adult male guinea pigs were used either as controls or immunized with Freund's complete adjuvant alone or together with pertussis vaccine. An additional group comprised animals immunized with a suspension of isologous spermatozoa emulsified in Freund's complete adjuvant and with pertussis vaccine. Ultrastructural studies of the testes of experimental animals showed, at earlier periods, apparently normal Sertoli junctions. However, in the adluminal compartment, distended gaps were seen between the facing membranes of adjacent Sertoli cells. At later periods, a massive destruction of the germinal cells were observed. In freeze-fracture replicas, the Sertoli junctions of testes belonging to all the experimental groups were characterized by an irregular network of occasionally interrupted strands of particles associated with the P face (PF). Large concavities determined distensions between interconnecting ridges. The gap junctions were increased in number and in surface. Tracer studies using horseradish peroxidase showed that the marker permeated the myoid cells of a greater proportion of tubules than in control animals. Within the seminiferous epithelium there was only a limited passage of the marker towards the lumina of the tubules. Yet the tracer was always excluded from the adluminal compartment by the Sertoli tight junctions. Our observations suggest the possibility that the FCA causes a loosening of the Sertoli junctions. This condition could enhance exchanges between two antigenically different cellular compartments and, thus, favor occurrence of an autoimmune reaction when cytotoxic factors are experimentally induced, as in iso- or autoimmunization.     

Multilayered structure of the basal lamina of the tubuli recti in normal mice

Previous studies have demonstrated that the blood-testis barrier (BTB) at the tubuli recti (TR) and the rete testis (RT) is less complete than at the seminiferous tubules (ST). However, there has been no report focusing on the basal lamina, which is an important component of the BTB at both TR and RT. In the present study, we performed electron microscopic observation of the basal lamina at the TR and RT, in comparison with that of those of the ST in normal mice. The results showed that the basal lamina of modified Sertoli cells at the TR segment exhibited a wavy and multilayered structure, but the Sertoli cells of ST and the epithelium of RT had an almost flat and singlelayered basal lamina. It was also noted that wide gaps existed between the modified Sertoli cells, the basal lamina of the epithelial layer, and the myoid cell layer at the middle TR segment. This characteristic structure of the basal lamina of the TR epithelial layer may be one of the factors for its incomplete BTB.     

Correlation of the presence of blood-brain barrier tight junctions and expression of zonula occludens protein ZO-1 in vitro: a freeze-fracture and immunofluorescence study

Tight junctions are regarded as the primary anatomical structure responsible for the blood-brain barrier (BBB). The molecular components that have been defined include ZO-1, a peripheral membrane protein associated with the cytoplasmic surface of the tight junction in epithelial and endothelial cells. It has been localized to the points of membrane contact with the fibrils seen by freeze-fracture. Examination of passaged endothelial cells with freeze-fracture failed to locate the intramembrane specializations associated with tight junctions. For this reason, immunocytochemistry and freeze-fracture were used to study the correlation of ZO-1 expression with the presence of tight junctions in bovine brain and aorta endothelial cells. Indirect immunofluorescence analysis showed ZO-1 to be localized at sites of cell-cell contact. Images of freeze-fractured sites of endothelial cell-cell contacts in identical passage numbers did not display characteristic tight junctions. When bovine aorta endothelial cells were cultured in astrocyte-conditioned medium on a complete extracellular matrix, platinum replicas displayed profiles of tight junctions. The elements of tight junctions were arranged as parallel ridges which displayed free ends. The immunofluorescence staining of ZO-1 was identical to that obtained on the endothelial cells that displayed no tight junction profiles. These results suggest that ZO-1 may be present at putative junction-containing sites before the junctional structures appear in the surface membrane. Therefore, ZO-1 expression does not a priori reflect assembly of the tight junctions identified by freeze-fracture.     

Overcoming barriers in the study of tight junction functions: from occludin to claudin

Tight junctions (TJs) are essential structures for the physiological functions of epithelial and endothelial cells, and have been suggested to have both barrier and fence functions. Tight junctions create a primary barrier to the diffusion of solutes through the paracellular pathway, and also function as a fence between apical and basolateral membrane domains, to create and maintain cell polarity of epithelial and endothelial cells. Several peripheral membrane proteins have been shown to be concentrated at the cytoplasmic surface of TJs. However, TJ-specific integral membrane proteins had not been identified until recently, and the lack of information concerning TJ-specific integral membrane proteins has hampered a more direct assessment of the function of TJs at the molecular level. Here, we present an overview of current progress in the identification of TJ-specific integral membrane proteins.     

The blood-testis barrier in experimental unilateral cryptorchidism

Unilateral cryptorchidism was induced in Wistar rat pups within 48 h of birth. After a period of 150 days, the blood-testis barrier was evaluated in the cryptorchid and contralateral scrotal testis, using a lanthanum immersion technique. The barrier was demonstrated to be competent, with tracer confined to the basal and intermediate compartments.     

Development of Endothelial Paracellular Clefts and their Tight Junctions in the Pial Microvessels of the Rat

The microvessels of the pia mater lack an investment with astrocyte processes but nonetheless have a high transendothelial electrical resistance which has caused them to be regarded as part of the blood-brain barrier. This high resistance is known to be acquired in the perinatal period. The aim of our study was to relate the known physiological changes with differentiation of the endothelial paracellular clefts and especially of their tight junctions which provide the basis for the high transendothelial resistance of blood-brain barrier vessels. Tight junctions of endothelial cell paracellular clefts in pial microvessels were examined by transmission electron microscopy using goniometric tilting to reveal and measure membrane separations at tight junctions in fetal, postnatal and adult rats. These tight junctional membrane separations narrowed over the period (E16:6.3 nm, D1:6.4 nm, D7:5.4 nm) and differentiated into two groups by the adult stage: one with a membrane separation of 2.8 nm and the staining characteristics of non-brain endothelial junctions, and the other with no detectable membrane separation and the staining characteristic of blood-brain barrier endothelial junctions. This patchy and incomplete differentiation of pial tight junctions into a blood-brain barrier-like form could result either from non-uniform exposure to inductive signals or to local variation in responsiveness to such agents. Although these changes in junction organization may be related to the known increase in pial transendothelial resistance in the perinatal period, we have not yet identified any sharply defined structural change which coincides with this physiological event.