Gap junctions between sertoli and germ cells of rat seminiferous tubules

Ultrastructural observations of rat seminiferous tubules show clearly the presence of plasma membrane junctions between Sertoli and germ cells in the basal and adluminal compartments. Results obtained from the freeze fracture and thin section techniques were correlated in order to elucidate the nature of these intercellular junctions. We suggest that these intercellular membrane specializations are gap junctions which occur within regions of plasma membrane that also exhibit adherens-like modifications.     

DHT deficiency perturbs the integrity of the rat seminiferous epithelium by disrupting tight and adherens junctions

In rats with a DHT deficiency induced by finasteride, morphological changes in the seminiferous epithelium were observed. The structural alterations were manifested by the premature germ cells sloughing into the lumen of seminiferous tubules. The etiology of this disorder could be connected with intercellular junctions disintegration. We showed in the immunohistochemical study the changes in expression of some proteins building tight and adherens junctions. The depression of N-cadherin, β-catenin and occludin immunoexpressions could be the reason for the release of immature germ cells from the seminiferous epithelium. However, the observed increase of the immunohistochemical reaction intensity of vinculin, one of the cadherin/catenin complex regulators, could be insufficient to maintain the proper function of adherens junctions. The hormonal imbalance appears to influence the pattern of expression of junctional proteins in the seminiferous epithelium. It could lead to untimely germ cells sloughing, and ultimately could impair fertility.     

Intercellular communication in rat seminiferous tubules

Intercellular electrical coupling in seminiferous tubules from prepubescent and adult Wistar rats has been studied by using conventional techniques. It is found that cells in the seminiferous epithelium are electrically coupled. Experiments performed using "Sertoli cell-enriched" seminiferous tubules indicate the existence of intercellular ionic communication between Sertoli cells. Junctional conductance is independent of the direction of electrical field and it is affected by A23187 Ca ionophore (5 microM) but not by exposure to the neurotransmitter norepinephrine (1-5 X 10(-5) M). Intracellular resistivity (including junctional resistance) is higher in mature as compared to immature germinal epithelium. These findings suggest that cell metabolites or second messenger molecules could be transferred via the low-resistance pathways between epithelium cells to coordinate cellular activity.     

Evidence that tubulobulbar complexes in the seminiferous epithelium are involved with internalization of adhesion junctions

Tubulobulbar complexes may be part of the mechanism by which intercellular adhesion junctions are internalized by Sertoli cells during sperm release. These complexes develop in regions where Sertoli cells are attached to adjacent cells by intercellular adhesion junctions termed ectoplasmic specializations. At sites where Sertoli cells are attached to spermatid heads, tubulobulbar complexes consist of fingerlike processes of the spermatid plasma membrane, corresponding invaginations of the Sertoli cell plasma membrane, and a surrounding cuff of modified Sertoli cell cytoplasm. At the terminal ends of the complexes occur clusters of vesicles. Here we show that tubulobulbar complexes develop in regions previously occupied by ectoplasmic specializations and that the structures share similar molecular components. In addition, the adhesion molecules nectin 2 and nectin 3, found in the Sertoli cell and spermatid plasma membranes, respectively, are concentrated at the distal ends of tubulobulbar complexes. We also demonstrate that double membrane bounded vesicles are associated with the ends of tubulobulbar complexes and nectin 3 is present on spermatids, but is absent from spermatozoa released from the epithelium. These results are consistent with the conclusion that Sertoli cell and spermatid membrane adhesion domains are internalized together by tubulobulbar complexes. PKCalpha, a kinase associated with endocytosis of adhesion domains in other systems, is concentrated at tubulobulbar complexes, and antibodies to endosomal and lysosomal (LAMP1, SGP1) markers label the cluster of vesicles associated with the ends of tubulobulbar complexes. Our results are consistent with the conclusion that tubulobulbar complexes are involved with the disassembly of ectoplasmic specializations and with the internalization of intercellular membrane adhesion domains during sperm release.     

Gap junctions and connexin-mediated communication in the immune system

Gap junctions and connexins are present in the immune system. In haematopoiesis, connexin 43, the most widely distributed gap junction protein, appears to be a key player in the development of progenitor cells and their communication with stromal cells. Connexin 43 is expressed by macrophages, neutrophils and mast cells. Lymphocytes also express connexin 43, and inhibition of gap junction channels in these cells by using highly specific connexin mimetic reagents has profound effects on immunoglobulin secretion and synthesis of cytokines. Lymphocytes and leukocytes also communicate directly in vitro with endothelial cells via gap junctions. Connexins are implicated in inflammatory reactions in a range of tissues. Their involvement in atherosclerotic plaque formation in the vascular system is also a current growth point in research, and could lead to the development of therapeutic interventions.     

Gap junctions and impulse propagation in embryonic epithelium of Amphibia

Summary Epithelium of amphibian embryos (Cynops orientalis, Xenopus laevis) was found in preceding experiments to generate and conduct impulses during a limited stage (26–37) of development. In order to elucidate the structural basis of impulse propagation, epithelial cells of four stages were examined by the freeze-etching method: (I) before and (II) during acquisition of conductivity; (III) when propagation was fully established, and (IV) when it was no longer present. Only few gap junctions (GJ) of small size were found in groups I and IV. GJ in epithelia of group III were increased in number and size, and appeared morphologically coupled, i.e., with more loosely arranged connexons. The size of gap-junctional particles did not differ significantly between coupled and uncoupled stages. Zonulae occludentes seemed leaky in stage I, and tight in stages II–IV. Thus, the morphological characteristics of specialized junctions between non excitable cells correlated with the opening and closing of low resistance intercellular current pathways during embryonic development.Gap junctions in particular seem to form an essential link in the non-neural stimulus-response system, which may facilitate the mobility of the embryo during early phases of aquatic life before the reflex pathways have been established. Coupling and uncoupling of gap junctions may also play an important role in the regulation of cell differentiation and morphogenetic movement. The experimental model used in this study provides a useful tool for further investigations of structural correlates of gap junctional permeability under physiological conditions.     

The structure of tight junctions in the tracheal epithelium may not correlate with permeability

Summary To test the hypothesis that cigarette smoke produces changes in the morphology of tight junctions guinea pigs were exposed to cigarette smoke or air in a previously standardized fashion (Simani et al. 1974). Permeability is greatest one half hour following exposure to cigarette smoke (Hulbert et al. 1981). The animals were sacrificed at that time. The tracheal epithelium was studied using both thin-section and freeze-fracture techniques. A quantitative analysis of the organization and integrity of junctional complexes was performed for each animal. Organization was assessed by measuring and comparing areas delimited by PF fibers and EF furrows. PF fiber integrity was assessed by measuring uninterrupted lengths of fibers and furrows from freeze-fracture replicas. This assessment did not demonstrate a change in tight-junction morphology following exposure to cigarette smoke.     

Blockage of testicular connexins induced apoptosis in rat seminiferous epithelium

Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of gap junction proteins, such as the connexin family members. Perturbation of their expressions may lead to spermatogenic arrest as manifested by disruption of cell-cell interaction. To explore the role(s) of connexins during spermatogenesis, we utilized the small peptide antagonistic approach to specifically deplete connexin 31, connexin 33, and pan-connexin. Three connexin peptides corresponding to the extracellular binding domain of connexin 31 and connexin 33 and to the extracellular conserved domain of connexins were designed and synthesized commercially. Peptides (at single dosage of 0.5, 1, or 2 mg per animal) were injected into rat testes and testes were collected on day 0, 1, 3, 5, 10, 15, and 30 after microinjection. In situ TUNEL assay demonstrated the induction of apoptosis in the testes after pan-connexin peptide treatment in a dose-dependent manner from day 3 and onward. Unlike the pan-connexin peptide, connexin 31 and connexin 33 peptides appeared to have little effect on inducing apoptosis and germ cell loss. CD45 staining also detected the occasional presence of infiltrating lymphocytes in the seminiferous tubules. Accompanied with the apoptotic events, two apoptotic markers, NF-κB and caspase 3, demonstrated a general up-regulation in their expressions. In adjacent testis sections, eliminations of connexin 31, 32, and 43 were observed. However, an induction of connexin 33 expression was detected. This suggests the versatility and functional diversity of connexins in the testis. The expression of ZO-1, the only known adaptor of connexins in the testis, was reduced and remained in a low level in the seminiferous epithelium. As such, the alterations of connexins in seminiferous epithelium may induce apoptotic signaling in the testis via the caspase 3 and the NF-κB pathway. This demonstrates the significant role of testicular connexins to maintain the survival of germ cells by regulating inter-cellular communications among germ cells and adjacent supporting cells during spermatogenesis. In addition, the inter-relationship between connexins and other junction proteins and associated signaling protein were investigated. After pan-connexin peptide treatment, a dys-localization of N-cadherin, an adherens junction protein, and diminution of occludin, a tight junction protein, level were detected. In addition, inductions of junction regulatory protein, cathepsin L, was observed during the course of peptide-mediated germ cell loss in the testes. In summary, pan-connexin peptide treatment triggered apoptosis and germ cell loss in the testes. This event influenced the localization and expression of different junction proteins and junction-associated protein in the testes. Financial support: The work was funded by a grant from the Research Grants Council of Hong Kong (HKU 7272/01M).     

Short-time effects of taxol on the seminiferous epithelium of the rat

Summary The effect of taxol, an inhibitor of microtubule degradation, on the seminiferous epithelium was studied. Taxol arrested spermatogenesis at metaphase in both mitotic and meiotic germ cell division. Microtubules were seen to accumulate, especially in the cytoplasm of the spermatogonia, and also in the early spermatids and Sertoli cells. No microtubule accumulation was observed in germ cells during meiotic prophase. Formation of the flagellum was affected in developing spermatids. Peculiar lamellar structures, probably derived from degenerating mitochondria, were seen in the cytoplasm of late spermatids and Sertoli cells.The results are compared with the effects of other mitotic inhibitors such as colchicine and vinca alcaloids.     

Microtubule depolymerization in rat seminiferous epithelium is associated with diminished tyrosination of alpha-tubulin

In the testis, microtubule-disrupting agents cause breakdown of the Sertoli cell cytoskeleton and sloughing of germ cells with associated Sertoli cell fragments, although the mechanism underlying this event is not understood. In this study, we investigated the effects of carbendazim and colchicine on microtubule polymerization status and posttranslational modifications of tubulin in freshly isolated rat seminiferous tubules. Soluble and polymerized tubulin pools were separated and tubulin was quantified using a competitive ELISA. Carbendazim and colchicine caused extensive microtubule depolymerization, shifting the ratio of soluble to polymerized tubulin from 40%:60% to 78%:22%, and to 84%:16%, respectively. Total tubulin levels remained relatively constant after carbendazim treatment but decreased twofold after colchicine treatment. To determine if modifications to tubulin may be associated with polymerization status, tubulin pools were analyzed by immunoblotting. Acetylated alpha-tubulin and betaIII-tubulin distribution in tubulin pools was not affected by treatment. Tyrosinated alpha-tubulin (52 kDa) was localized in both tubulin pools and had decreased tyrosination in the microtubule pool after carbendazim treatment. A 47-kDa protein immunoreactive with both tyrosinated alpha-tubulin and general alpha-tubulin antibodies was found only in the microtubule pool. The 47-kDa protein (potentially an alpha-tubulin isoform) lost tyrosination, yet was still present in the microtubule pool based on detection with the general alpha-tubulin antibody, after carbendazim treatment. Similar effects were seen with colchicine, although loss of total tubulin protein was measured. Thus, decreased tyrosination of the microtubule pool of tubulin appears to be associated with depolymerization of microtubules.     

Immunohistochemical localization of the transferrin receptor in the seminiferous epithelium of the rat

The transferrin receptor has been immunohistochemically localized in the seminiferous epithelium of the rat with a monoclonal antibody, MRC OX26, which recognizes the transferrin receptor glycoprotein. The receptor was detectable on mitotically and meiotically dividing germ cells and, less abundantly, on round spermatids. It was lost from germ cells during spermatid elongation and was undetectable on immature spermatozoa. The transferrin receptor was also present on Sertoli cells in the testes of immature animals and on Sertoli cells in the testes of aspermatogenic animals that had been irradiated in utero. It was not detectable on Sertoli cells in the testes of cryptorchid animals. These studies demonstrate that the transferrin receptor is abundant on dividing germ cells as well as dividing somatic cells.     

Sertoli cell ectoplasmic specializations in the seminiferous epithelium of the testosterone-suppressed adult rat

The Sertoli cell ectoplasmic specialization is a unique junctional structure involved in the interaction between elongating spermatids and Sertoli cells. We have previously shown that suppression of testicular testosterone in adult rats by low-dose testosterone and estradiol (TE) treatment causes the premature detachment of step 8 round spermatids from the Sertoli cell. Because these detaching round spermatids would normally associate with the Sertoli cell via the ectoplasmic specialization, we hypothesized that ectoplasmic specializations would be absent in the seminiferous epithelium of TE-treated rats, and the lack of this junction would cause round spermatids to detach. In this study, we investigated Sertoli cell ectoplasmic specializations in normal and TE-treated rat testis using electron microscopy and localization of known ectoplasmic specialization-associated proteins (espin, actin, and vinculin) by immunocytochemistry and confocal microscopy. In TE-treated rats where round spermatid detachment was occurring, ectoplasmic specializations of normal morphology were observed opposite the remaining step 8 spermatids in the epithelium and, importantly, in the adluminal Sertoli cell cytoplasm during and after round spermatid detachment. When higher doses of testosterone were administered to promote the reattachment of all step 8 round spermatids, newly elongating spermatids associated with ectoplasmic specialization proteins within 2 days. We concluded that the Sertoli cell ectoplasmic specialization structure is qualitatively normal in TE-treated rats, and thus the absence of this structure is unlikely to be the cause of round spermatid detachment. We suggest that defects in adhesion molecules between round spermatids and Sertoli cells are likely to be involved in the testosterone-dependent detachment of round spermatids from the seminiferous epithelium.     

Effects of spaceflight on the spermatogonial population of rat seminiferous epithelium

Testes from rats flown on Cosmos 1887 were compared with vivarium control and synchronous control samples. The mean weights of flight testes, normalized for weight per 100 g, were 6.4% less when compared with the vivarium controls. Counts of spermatogonia from tissue sections (seminiferous tubules in maturation stage 6) from five animals in each group revealed 4% fewer spermatogonia in flight testes compared with synchronous controls and 11% fewer spermatogonia in flight samples compared with vivarium controls.     

Frequency of the stages in the cycle of the seminiferous epithelium in the rat

This study determined the optimum number of tubules to be counted per testis cross section, and the number of animals per treatment group, when changes in stage frequencies in the cycle of the seminiferous epithelium are criteria for assessing effects of treatment on spermatogenesis. A data base of 9,672 observed and staged tubules was collected from testicular cross sections of 15 Sprague-Dawley rats. A significant variation between animals was found for the frequencies of Stages I, II, IV, VI, VIII, and XIII. Computer simulation was used to randomly select different combinations of animal and tubule numbers from the observed data. Stage frequency means from each simulation experiment were compared statistically to observed mean frequencies. A model that used data from all 14 stages was analyzed. The following conclusions were made: a) a minimum of 200 tubule cross sections/testis is recommended for estimating stage frequencies; b) for a fixed number of tubules scored, the number of animals sampled is more important than the number of tubules per animal in reducing variance; c) to detect a difference of 2 standard deviations from the mean with a 2% error rate and examining 200 tubules/testis, at least 12 animals must be used per group when assessing all 14 stages; d) when individual stages are examined using 10 animals per group, only Stage VII has 80% or greater power of test (alpha = 0.05) to detect a frequency difference; e) pooling stages into 3-4 groups is recommended to improve the power of detecting a treatment difference.     

Adjudin-mediated junction restructuring in the seminiferous epithelium leads to displacement of soluble guanylate cyclase from adherens junctions

A plethora of evidence supports the role of cyclic nucleotides in junction restructuring. For instance, studies have shown cGMP to be a key regulator of junction assembly and disassembly in different in vitro and in vivo systems. In this study, we examine the role of soluble guanylate cyclase (sGC) in junction restructuring in the seminiferous epithelium of the rat testis. First, the interaction of soluble guanylate cyclase beta1 (sGCbeta1; sGC is a heterodimer comprised of an alpha and a beta subunit) with proteins that constitute adherens and tight junctions in the testis was demonstrated. By immunoprecipitation, sGCbeta1 was found to associate with occludin, JAM-A, and ZO-1, as well as with cadherin, catenin, nectin, afadin, ponsin, and espin, suggestive of its role in cell junction dynamics. These results were corroborated in part by immunohistochemistry experiments, which revealed that the localization of sGCbeta1 was largely restricted to the site of the apical and basal ectoplasmic specialization. Next, the role of sGC in junction dynamics was addressed by using an in vivo model of junction restructuring. Administration of Adjudin--a chemical entity known to specifically perturb adhesion between Sertoli and germ cells (i.e., round and elongate(ing) spermatids and most spermatocytes)--resulted in a approximately 1.5-fold increase in sGCbeta1, coinciding with the loss of germ cells from the epithelium. More importantly, the ability of sGCbeta1 to associate with cadherin increased approximately three-fold during Adjudin-mediated restructuring of Sertoli-germ cell junctions, whereas its interaction with tight junction proteins (i.e., occludin and ZO-1) decreased. Taken collectively, these results suggest that sGC participates in the remodeling of cell junctions during spermatogenesis.