Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad.

In mammals a single gene on the Y chromosome, Sry, controls testis formation. One of the earliest effects of Sry expression is the induction of somatic cell migration from the mesonephros into the XY gonad. Here we show that mesonephric cells are required for cord formation and male-specific gene expression in XY gonads in a stage-specific manner. Culturing XX gonads with an XY gonad at their surface, as a 'sandwich', resulted in cell migration into the XX tissue. Analysis of sandwich gonads revealed that in the presence of migrating cells, XX gonads organized cord structures and acquired male-specific gene expression patterns. From these results, we conclude that mesonephric cell migration plays a critical role in the formation of testis cords and the differentiation of XY versus XX cell types.     

Sertoli cell behaviors in developing testis cords and postnatal seminiferous tubules of the mouse

Sertoli cells are the primary structural component of the fetal testis cords and postnatal seminiferous tubules. Live imaging technologies facilitate the visualization of cell morphologies and behaviors through developmental processes. A transgenic mouse line was generated using a fragment of the rat Gata4 gene to direct the expression of a dual-color fluorescent protein reporter in fetal and adult Sertoli cells. The reporter encoded a red fluorescent protein, monomeric Cherry (mCherry), fused to histone 2B and enhanced green fluorescent protein (EGFP) fused to a glycosylphosphatidylinositol sequence, with a self-cleaving 2A polypeptide separating the two fusion proteins. After translation, the red and green fluorescent proteins translocated to the nucleus and plasma membrane, respectively, of Sertoli cells. Transgene expression in testes was first detected by fluorescent microscopy around Embryonic Day 12.0. Sertoli cell division and migration were visualized during testis cord formation in organ culture. Initially, the Sertoli cells had mesenchyme-like morphologies and behaviors, but later, the cells migrated to the periphery of the testis cords to become epithelialized. In postnatal seminiferous tubules, Sertoli nuclei were evenly spaced when viewed from the external surface of tubules, and Sertoli cytoplasm and membranes were associated with germ cells basally in a rosette pattern. This mouse line was bred to previously described transgenic mouse lines expressing EGFP in Sertoli cytoplasm or a nuclear cyan fluorescent protein (Cerulean) and mCherry in plasma membranes of germ cells. This revealed the physical relationship between Sertoli and germ cells in developing testis cords and provided a novel perspective on Sertoli cell development.     

Quantitation of Sertoli cell-germ cell desmosome gap junctions in relation to meiotic divisions in the male rat.

Desmosome-gap (D-G) junctions were quantified in relation to germ cell meiosis in the male, specifically to test the hypothesis that the loss of these junctions is related to successful passage of cells through diplotene phase of Meiosis I and the two cytokineses that follow. Such a hypothesis has been proposed as the cause for the resumption of meiosis that occurs prior to ovulation in the female. D-G junctions were quantified in pachytene spermatocytes (stage XII), diplotene spermatocytes (stage XII), secondary spermatocytes (stage XIV) and step 1 spermatids (stage I). These were referred to as the cells of interest as compared with spermatocytes (zygotene spermatocytes, zygotene spermatocytes, pachytene spermatocytes, pachytene spermatocytes) in the same stages, respectively, that served as controls termed control cells. Since gap junctions are not easily recognized in the average sectioned profile of a desmosome-gap junction, only the desmosomal component was quantified. The data were expressed as both numbers and length of junctions per tubule, per cell profile and per unit lineal membrane length to overcome errors inherent in the methodologies utilized. There was no indication that numbers of junctions changed specifically in the cells of interest after passage through diplotene suggesting that these junctions do not have a comparable role in meiotic continuance in the male as proposed for the female. Interestingly, the control cells always showed greater numbers and length of junctions than the cells of interest suggesting that junction may relate more to the period of initiation of meiosis than to its continuance.     

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.     

Acetylcholine receptor distribution on regenerating mammalian muscle fibers at sites of mature and developing nerve-muscle junctions

When rat soleus muscles fibers regenerated after notexin-induced damage, AChRs were present at high density on the surface of the new muscle fibers at the sites of the original NMJs, even if the intact motor axons were not present during regeneration. Some AChR molecules which were labelled with R-BgTx before notexin-induced damage persisted for some days at junctional sites after new muscle fibres had regenerated. During muscle fiber degeneration, components of the muscle fiber plasma membrane appeared to remain longer in the junctional region than elsewhere. When muscles on which new "ectopic" NMJs had been forming for at least 2 weeks were damaged, AChR clusters together with sites of high AChE activity were present 2 weeks later on the regenerated muscles in the region of new NMJ formation, even if the "foreign" nerve was not intact during the period of regeneration. If ectopic NMJs had been forming for only 4 days at the time of muscle and nerve damage, neither AChR clusters nor AChE activity were detected on the regenerated muscle fibers.     

Association between mitochondria and gap junctions in mammalian myocardial cells

In ventricular myocardial cells of mouse, guinea-pig, dog, and monkey, mitochondria frequently form close associations with gap junctions, the two structures being separated by a space of 20 nm or less. Similar appositions are found in both the mature atria and the developing myocardium of the mouse. The gap junctions assume a variety of configurations with respect to the apposed mitochondria. These include profiles in which the gap junctions conform closely to the contours of mitochondria, as well as profiles in which finger-like sarcolemmal evaginations, composed entirely of gap junctions, extend longitudinally or transversely into an adjoining cell to envelop mitochondria. In mouse ventricular wall, over 40% of the length of gap junctions is juxtaposed to mitochondria, and strands of connecting material are often present in the interspace between the two structures. In addition, in freeze-fracture replicas, portions of mitochondria are found attached to areas of myocardial sarcolemma that contain gap-junctional particles. Since mitochondria are known to sequester Ca²⁺ ion, it is possible that the close association between mitochondria and gap junction may function to buffer the intracellular Ca²⁺ concentration near the gap junctions, and thereby regulate the ionic permeability of the gap junctions.     

Role of FSH and triiodothyronine in Sertoli cell development expressed by formation of connexin 43-based gap junctions

Follicle-stimulating hormone (FSH) and triiodothyronine (T3) are known regulatory factors of spermatogenesis initiation. Connexin 43 (Cx43) is the most ubiquitous constitutive protein of gap junctions in the testis. This study evaluates the effects of the hyperstimulation of FSH and T3 during testicular maturation on Cx43 expression in the testis. The newborn, male Wistar rats were divided randomly into four experimental groups: FSH group-daily injections of FSH 7.5?IU/animal; T3 group-100?μg T3/kg body weight; FSH+T3 group-both substances; A control group-received vehicles in the same volume. Proliferating cell nuclear antigen immunohistochemistry and toluidine blue staining were used to determine the germ cell proliferation and degeneration. Cx43 immunolocalization was evaluated to find Cx43 maturational changes. Under FSH treatment, the proliferation rate was high so the total number of Sertoli cells increased with a low level of degeneration and lumen formation. T3 stimulation evoked a reduction in the proliferation rate and a decrease in Sertoli cell number but with intensive formation of lumen. T3+FSH inhibited the proliferation rate and stimulated lumen formation together with degeneration, which negatively influenced the number of germ cells in the seminiferous epithelium. We conclude that T3 action seems to be particularly connected with the maturation of Cx43 gap junctions. FSH stimulates maturation of Sertoli cell function, but this effect may take place regardless of the presence of Cx43-dependent intercellular communication. The hyperstimulation of both FSH and T3 damages Cx43 connections and hence evokes regressional changes in the seminiferous epithelium.     

Structure and function of gap junctions in the developing brain

Gap-junction-dependent neuronal communication is widespread in the developing brain, and the prevalence of gap-junctional coupling is well correlated with specific developmental events. We summarize here our current knowledge of the contribution of gap junctions to brain development and propose that they carry out this role by taking advantage of the full complement of their functional properties. Thus, hemichannel activation may represent a key step in the initiation of Ca²⁺ waves that coordinate cell cycle events during early prenatal neurogenesis, whereas both hemichannels and/or gap junctions may control the division and migration of cohorts of precusor cells during late prenatal neurogenesis. Finally, the recent discovery that pannexins, a novel group of proteins prominently expressed in the brain, are able to form both hemichannels and gap-junction channels suggests that we need to seek more than just connexins with respect to these junctions.Work in the authors’ laboratories is supported by the Deutsche Forschungsgemeinschaft, SFB 509 (R.D.) and by the Institut Pasteur (R.B.).     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.     

Biochemical and genetic investigations on gap junctions from mammalian cells

Gap junction protein (26K) in mouse or rat liver has been studied using a rabbit antiserum directed against the sodium dodecylsulfate denatured 26K protein from mouse liver. The liver 26K protein has been localized in gap junction plaques of hepatic plasma membranes by immuno electron microscopy. Affinity purified anti-26K antiserum showed weak cross reactivity with mouse or bovine lens gap junction protein (MIP26). This result suggests some structural homology between the different gap junction proteins in liver and lens. After partial hepatectomy of young rats the liver 26K protein appears to be degraded and later resynthesized. A variant of established Chinese hamster fibroblastoid cells has been isolated and shown to be defective in metabolic cooperation via gap junctions.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Development of Sertoli cell junctions in vitro--a freeze-fracture study

Seminiferous tubules of 1-day-old rats were maintained in organ culture for up to 40 days. Five classes of intercellular junctions between Sertoli cells were observed by the freeze-fracture method as the tissue aged: (a) typical gap junctions; (b) focal tight junctions; (c) macular tight junctions; (d) meandering tight junctions; and (e) extensive tight junctions. The relative proportions of these types of Sertoli cell junctions were quantitated as the organ cultures progressed. The junctional structures observed and classified in organ culture were identical to those seen in vivo, but the timing of their appearance and/or disappearance, as well as their relative proportions, was different from that observed in the developing animal. Extensive tight junctions, with numerous parallel strands, were observed in the 40-day cultures; however, their oblique orientation with respect to the myoid layer was in contrast to the parallel orientation observed in vivo.     

Claudins in occluding junctions of humans and flies

The epithelial barrier is fundamental to the physiology of most metazoan organ systems. Occluding junctions, including vertebrate tight junctions and invertebrate septate junctions, contribute to the epithelial barrier function by restricting free diffusion of solutes through the paracellular route. The recent identification and characterization of claudins, which are tight junction-associated adhesion molecules, gives insight into the molecular architecture of tight junctions and their barrier-forming mechanism in vertebrates. Mice lacking the expression of various claudins, and human hereditary diseases with claudin mutations, have revealed that the claudin-based barrier function of tight junctions is indispensable in vivo. Interestingly, claudin-like molecules have recently been identified in septate junctions of Drosophila. Here, we present an overview of recent progress in claudin studies conducted in mammals and flies.     

Freeze-fracture studies of gap junctions in the developing and adult amphibian cardiac muscle.

The freeze-fracture technique has been used to characterize the junctional devices involved in the electrical coupling of Ambystoma cardiac tissue. These cells are connected by junctions formed by either linear or circular arrays of particles. Such structures can be interpreted as a special type of gap junction. Gap junctions have also been investigated during the growth and differentiation of two amphibians, Rana and Xenopus. In both genera the earliest stage of junctional assembly is characterized by linear rows of particles. Later, a gradual transformation of these linear rows into circles was found. Finally, in the fully formed gap junctions, these circles appeared to join together into clusters. In summary, in the adult amphibian myocardial cells, three different types of gap junctions can be described. The first type, which has been observed in all embryonic stages and in adults in all three genera, consists of linear or circular arrays of particles: this is the only type of gap junction seen at any age in Xenopus. The second type, consisting of a variable number of anastomosing circles forming regular networks, is never observed in embryonic cells. It is typical of the adult frog heart and may also be seen in Ambystoma. The third type is characteristic only of adult Ambystoma heart and consists of geometrically packed particles identifiable with classic communicating macula. The fact that only the first class of structure is observed in Xenopus heart strongly supports the conclusion that such linear arrays of intramembranous particles really represent true functional electrical junctions.     

Sertoli cell expression of galectin-1 and -3 and accessible binding sites in normal human testis and Sertoli cell only-syndrome.

Galectins are vertebrate lectins interacting with beta-galactosides and derivates thereof such as blood group A, B and H determinants. The expression of gelectin-1 and -3 and galectin-specific binding sites by human Sertoli cells was analyzed in normal human testis and Sertoli cell only-syndrome (SCOS). Staining intensity was scored semiquantitatively on a 4-grade scale. Sertoli cells in normal testes displayed a moderate cytoplasmic and weak nuclear staining for galectin-1-specific binding sites. Galectin-3-specific binding sites were expressed in Sertoli cells less intensely than accessible ligands for galectin-1 (mean score 2.25 for galectin-1 and 1.50 for galectin-3). Germ cells were only weakly reactive. Tubular walls were negative for both classes of galectin-specific binding sites. In SCOS, galectin-1 binding was moderate to strong and more pronounced than galectin-3 binding by Sertoli cells (mean scores 4.00 and 2.25). Tubular walls were negative for galectin-staining. The ratio for galectin-1-/galectin-3-specific binding (staining score ratio) was 1.50 form normal testis and 1.78 for SCOS disclosing a relative increase of galectin-3 binding sites in the latter. Staining with galectin-1- and -3-specific antisera showed a strong cytoplasmic galectin-1 immunoreactivity in Sertoli cells of normal and SCOS testis (score 4.00 for both). Anti-galectin-3 did not stain Sertoli cells or germ cells in normal testis. Only Leydig cells were labeled (score 3.00). In SCOS a weak to moderate nuclear staining of Sertoli cells was noted (score 2.00). Galectin-3 expression and galectin-1-specific binding sites were found to be increased in Sertoli cells of SCOS. This modulation of reactivity can have implications for Sertoli cell interactions with galectin-reactive extracellular matrix components like laminin and for anti-apoptotic effects.     

Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis

Members of the newly identified claudin gene family constitute tight junction (TJ) strands, which play a pivotal role in compartmentalization in multicellular organisms. We identified oligodendrocyte-specific protein (OSP) as claudin-11, a new claudin family member, due to its sequence similarity to claudins as well as its ability to form TJ strands in transfected fibroblasts. Claudin-11/OSP mRNA was expressed in the brain and testis. Immunofluorescence microscopy with anti-claudin-11/OSP polyclonal antibody (pAb) and anti-neurofilament mAb revealed that in the brain claudin-11/OSP-positive linear structures run in a gentle spiral around neurofilament-positive axons. At the electron microscopic level, these linear structures were identified as the so-called interlamellar strands in myelin sheaths of oligodendrocytes. In testis, well-developed TJ strands of Sertoli cells were specifically labeled with anti-claudin-11/OSP pAb both at immunofluorescence and electron microscopic levels. These findings indicated that the interlamellar strands of oligodendrocyte myelin sheaths can be regarded as a variant of TJ strands found in many other epithelial cells, and that these strands share a specific claudin species, claudin-11/OSP, with those in Sertoli cells to create and maintain the repeated compartments around axons by oligodendrocytes.     

The occluding junctions of mouse duodenal enterocytes during development

Summary The architecture of occluding junctions during the differentiation of the mouse duodenum was studied in freeze-fractured material. Irregular zonulae occludentes (ZO) (Type I) are numerous during fetal life, and are characterized by their irregular width, and by the presence of basal open-ended extensions fused with the discontinuous basal strand of the ZO. Regular ZOs (Type II), typical of the adult villous epithelium, appear after Type I junctions by day 16 of gestation. Two patterns are distinguishable: in the first, parallel strands of ridges and furrows are found without crossing branches; in the second pattern, the junction zone is organized like a network of short branches forming various types of polygons. In fetal and adult mice fasciae occludentes (FO) (Type III) are present on the lateral cell membranes; in unfixed specimens particles are found in the furrows of the E-face and pits on the ridges of the P-face. In fixed tissues, the particles are aligned on the ridges of the P-face. These results indicate that fixation with glutaraldehyde modifies considerably the affinity of junctional particles toward the P-face during the fracture process. Moreover, the presence of numerous large FOs on the lateral cell membranes of enterocytes during late fetal life and in the adult, is possibly related to cell movement along the intestinal villi.