Fumie Suzuki

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

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

Development of tight junctions in the caput epididymal epithelium of the mouse.

Abstract The course of development of the epithelial tight junctions of the Wolffian duct and the caput epididymal principal cells in the mouse were examined by freeze-fracture. The histogenesis of the epididymis is briefly described. In the 12-day embryo, tight junction meshworks surround the entire circumference of the columnar cells in the juxtaluminal position. During fetal life, the strands are more discontinuous than those of postnatal mice, and two or more strands frequently run together. Up to 10 days of age, the basal compartments of the tight junctions are much larger than the luminal ones. Marked increases in both the number of strands and the depth of the tight junctions appear by 20 days. Strands with a terminal loop are often observed up to 16 days, except for the newborn stage, suggesting that the formation of the terminal loop is related to the active elongation of the strands. The tight junctions increase greatly in number and depth near three-cell junctions. Up to 20 days, the strands anastomose frequently, with no particular orientation to the cell axis. After 20 to 37 days, the direction of the strands becomes parallel to the luminal surface, with a decreased number of anastomoses as the lumen widens. In the adult, the number of sealing strands is about 10 within the depth of the tight junctions. Free-ended strands are seen in all stages examined. The formation of the tight junction meshworks is discussed in the light of the findings during the development.

Morphogenesis of tight junctions in the peritoneal mesothelium of the mouse embryo.

SummaryThe peritoneal mesothelium of mouse embryos (12 to 18 day of gestation) was studied by freeze-fracture and in sections in order to reveal the initial formation of the tight junctions. Freeze-fracture observations showed three types of tight junctions. Type I consists of belt-like meshworks of elevations on the P face and of shallow grooves on the E face. No tight junctional particle can be seen either on the elevations or in the grooves. Type II shows rows of discontinuous particles on the elevations on the P face. Type III consists of strands forming ridges on the P face. On the E face, the grooves of Type II and III appear to be narrower and sharper than those of Type I. Quantitatively, Type I junctions are most numerous during the early stages (day 12–13) of embryonic development, while Type III junctions become more common in the later stages, and are the only type seen by day 18. Observations on sections, however, fail to distinguish between the three types.The results suggest that an initial sign of tight junction formation is close apposition of the two cell membranes in the junctional domain, without tight junctional particles. Later, the particles appear to be incorporated in the tight junctions and the strands form by fusion of the particles.

Changes in the distribution of intramembranous particles and filipin-sterol complexes during epididymal maturation of golden hamster spermatozoa

Golden hamster spermatozoa in various segments of the excurrent duct system were studied by freeze-fracture with and without filipin treatment. Two types of regular IMP (intramembranous particle) patterns temporarily appear on the plasma membrane covering the sperm head. One is a hexagonal arrangement seen in the acrosomal region, and the other is a linear arrangement near the posterior ring. Both patterns are seen in the spermatozoa from the corpus epididymidis. The FSC (filipin-sterol complex) density in the plasma membrane covering the acrosome increases from about 400 to 500 FSC/microns2 during epididymal passage. In this region, the majority of the membrane sterols appears to reside on the outer leaflet of the lipid bilayer. When the spermatozoa reach the cauda epididymidis, FSCs in the outer acrosomal membrane virtually disappear from the apical segment, while they increase in the middle segment (250 FSC/microns2). These observations are discussed in relation to epididymal maturation.

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

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

Cell Junctions in the Seminiferous Tubule and the Excurrent Duct of the Testis: Freeze-Fracture Studies

Publisher Summary This chapter describes the morphological observations of intercellular junctions in the seminiferous tubule and the excurrent duct system of mammalian testes as revealed by freeze fracturing. The junctions in the nonmammalian testes are also described. The seminiferous epithelium resting on the seminiferous tubule consists of two cellular populations: spermatogenic cells and Sertoli cells. These originate from different germinal layers during histogenesis. All generations of the clonal spermatogenic cells are connected with each other by the cytoplasmic bridges to form syncytia until spermiation. The Sertoli cells or supporting cells never divide in the adult testis and are separated by their cell membranes as individual cells. Intercellular junctions among the epithelial lining cells other than the Sertoli cells are recognized as the terminal bar by light microscopy. The junctional complexes of the epithelial cells lining the excurrent duct system are situated in the juxtaluminal area of the cell. More comparative, experimental, and clinicopathological studies on the junctional complexes would be necessary to understand the blood–testis barrier and the environments of the excurrent duct system.

Membrane changes in Chinese hamster spermatozoa during epididymal maturation

Chinese hamster spermatozoa during epididymal maturation were examined by thin sectioning, freeze-fracture, and surface replica. Membrane-limited vesicles and tubules (MVTs) attach to the plasma membrane over the acrosome of the spermatozoa in the distal caput through proximal cauda epididymidis. The origin of these MVTs is likely to be spermatozoa degenerating in the epididymal lumen. The attachment of MVTs to the plasma membrane seems to be mediated by a paste-like substance covering the plasma membrane. A parallel striation pattern of intramembranous particles (IMPs) is seen in the plasma membrane of almost the entire postacrosomal region of the epididymal spermatozoa. The patterned domain begins to appear in the proximal caput epididymidis. The number and density of IMPs in the plasma membrane of the postacrosomal region increases with the development of striated pattern of IMPs in this region. In the cauda epididymidis, the redundant nuclear envelope elongates to form a shirt-like membrane covering the mitochondrial sheath. The elongated portion of the nuclear envelope is devoid of nuclear pores and has few IMPs.

Three-dimensional model of tight junction fibrils based on freeze-fracture images

SummaryTo study the three-dimensional structure of tight junction fibrils, the epithelia of the jejunum and epididymis of adult mice were examined by the freezefracture technique in unfixed and in aldehyde-fixed specimens. The fibrils have a stronger affinity for the protoplasmic (P) face of the lipid bilayer in fixed material, and for the external (E) face in unfixed and rapidly frozen material. Therefore we can observe the fibrils both from the outside and inside of the cell. Fibrils appearing on the P-face are smoothly contoured ridges and rows of hemispherical particles, while those appearing on the E-face are exclusively rows of hemispherical particles. Based on these observations, we wish to propose a new fibril model for the tight junction. There are two distinctive types of junctional elements. One type is composed of a smooth and continuous strand in the external view of the cell, but is studded with hemispherical bulgings in its internal view. This type will be referred to as the “continuous type”. The other type is bead-like, and will be referred to as the “particle type”. The relative proportion of these two types of elements appearing within a tight junction network differs among tissues.

Freeze-fracture observations of ovulated hamster oocytes with their cumulus cells

SummaryMature unfertilized oocytes in the cumulus oophorus were examined using the freeze-fracture technique. The P face of the oocyte plasma membrane (oolemma) has randomly distributed intramembranous particles (IMPs) with an average diameter of about 10 nm. No marked difference is found between the intermicrovillous and microvillous regions of the oocyte with respect to the size and distribution of IMPs. The E face of the oolemma has fewer IMPs than the P face. The intermicrovillous region is slightly elevated in some places because of close contact with underlying cortical granules. The oolemma of this elevated region and the membrane of the cortical granule have IMPs which are considerably smaller than those in the remaining areas of the oolemma. The IMP density of the P face of the cortical granule membrane is much lower than that of the E face, possibly indicating a membrane specialization required for fusion of the cortical granule membrane with the overlying oolemma at the time of exocytosis. Cytoplasmic lamellae appear as parallel bands of particles in freeze-fractured specimens. No gap junctions are found between the oocyte and cumulus cells, but residual gap junctions are seen among clustered cumulus cells.

Distribution of Intramembrane Particles and Its Changes during the Acrosome Reaction in Spermatozoa of the Japanese Abalone

The distribution of intramembrane particles in the plasma and acrosomal membranes of sperm of the Japanese abalone, Haliotis discus, and its changes during the acrosome reaction were studied by the freeze‐fracture replica technique. The P face of the plasma membrane covering the acrosome has sparse membrane particles except in the apical region, which includes the trigger and ‘truncated cone’ regions. Large particles with an average diameter of 10 nm are located in this apical region. The E face of the plasma membrane has only a few particles. On the outer acrosomal membrane, many particles are randomly distributed throughout the P face, but only a small number of particles are found on the E face. Numerous particles on the P face of the inner acrosomal membrane show a regular arrangement as a dense lattice or with a concentric circular pattern. The initial change in the acrosome reaction is clearance of membrane particles from both the P and E faces of the plasma and outer acrosomal membranes around the apical region, where fusion of the two membranes occurs. As the acrosomal process elongates, the dense arrangement of particles on the inner acrosomal membrane changes via a loose lattice arrangement to a patchy distribution with particle‐free areas. Then the arrangement is further disorganized becoming a sparse, random distribution.