Recently, a novel family of gap junction (GJ) proteins, the pannexins (Panxs), has been identified in vertebrates. To evaluate the role of Panxs in the male reproductive tract (MRT), we investigated the expression and distribution of Panx1 and Panx3 in the testis, efferent ducts (EDs), and epididymis of adult rats. In the testis, Panx1 localized to Sertoli cells in the basal compartment of seminiferous epithelium, while Panx3 was expressed in Leydig cells. In the EDs, both Panx1 and Panx3 were expressed in the epithelium, notably at the apical region of ciliated cells. In the epididymis, both Panxs were expressed in principal cells. Prominent labelling for Panx1 was also observed laterally and basally between adjacent principal cells and at the interface between principal and basal cells. Panx3 was noted apically in principal cells and at the plasma membrane of halo cells. RT-PCR and Western blots analyses confirmed the expression of both Panxs in the MRT, with multiple species being detected in western blots and observed to be expressed in a region-specific manner, corroborating previous reports of post-translational glycosylation of Panxs. Preliminary RT-PCR results suggested that Panx1 may be regulated at the mRNA level since alternative splicing may account for some of these species. Testosterone and other testicular factors appear to regulate expression of Panxs, as an increase in the number of Panx1 species and a change in the expression of Panx3 species were observed with orchidectomy, and these were partially inhibited by testosterone supplementation. In conclusion, Panxs are present throughout the MRT where they are expressed in a cell type- and region- specific manner and appear to be regulated in terms of expression levels, subcellular localization and post-translational modification. Taken together their wide spread distribution suggest an important role for Panxs in the MRT. / Une nouvelle famille de protéine de jonctions lacunaires, les pannexines (Panxs), a récemment été découverte chez plusieurs espèces, incluant chez les vertébrés. Pour évaluer le rôle des Panxs dans le système reproductif mâle (SRM), l'expression et la distribution de Panx1 et Panx3 ont été étudiées dans le testicule, les canaux efférents et l'épididyme de rats adultes. Dans le testicule, Panx1 était présente dans les cellules de Sertoli, dans la région basale des canaux séminifères, tandis que Panx3 était exprimée par les cellules de Leydig. Dans les canaux efférents, les deux Panxs étaient exprimées dans l'épithélium, principalement dans la région apicale des cellules ciliées. Finalement, dans l'épididyme, Panx1 et Panx3 étaient localisées dans les cellules principales. Panx1 se retrouvait dans la région latérale et basale entre les cellules principales adjacentes en plus d'être présente à l'interface entre les cellules principales et basales. Panx3 était présente dans la région apicale des cellules principales et à la membrane plasmique des cellules halo. Les analyses de RT-PCR et d'immunobuvardage de type Western ont confirmé la présence des deux Panxs dans le SRM. Ces analyses ont mis en évidence plusieurs bandes spécifiques pour chaque Panxs dans les différentes régions, ce qui corrobore avec de récentes évidences démontrant la glycosylation post-traductionnelle des Panxs. Des résultats préliminaires de RT-PCR démontrant un épissage alternatif de Panx1 suggère aussi une régulation au niveau de l'ARNm et certaines des bandes obtenues en immunobuvardage pourraient être dues à ce processus. La testostérone et d'autres facteurs testiculaires semblent jouer un rôle dans la régulation des Panxs, car une augmentation du nombre de bandes pour Panx1 et un changement dans l'expression des bandes pour Panx3 au niveau de la protéine ont été observés suite à la castration des animaux, et ces changement

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No description available.

The Na,K-ATPase is a ubiquitous plasma membrane enzyme which is responsible for the establishment of Na+ and K+ homeostasis in animal cells. In the small intestine, the establishment of an electrochemical Na+ gradient at the apical membrane of the villus absorptive cell is crucial for net nutrient and electrolyte absorption. Na,K-ATPase is made up of a large (112KDa) catalytic $ alpha$ subunit, and a glycosylated (35KDa) $ beta$ subunit. Thus far, 3 isoforms for both the $ alpha$ and $ beta$ subunit have been described. We sought to characterize both age-related changes and regional changes in levels of small intestinal Na-K ATPase activity and subunit isoform expression (except $ beta$3) during the postnatal development of the rat. In the proximal small intestine, the specific activity of Na,K-ATPase measured in subcellular fractions gradually rose, while in the distal segment, this rise was less marked. A proximal to distal gradient in Na,K-ATPase activity was demonstrated in post-weaned rats. Only $ alpha$1 and $ beta$1 subunit isoforms were detectable by Western blotting and their expression paralleled the age-specific and regional changes observed in the levels of Na,K-ATPase activity. In addition, the glycoprotein $ beta$1 demonstrated an increase in molecular weight during ontogenesis. The results of this study show important changes in the activity and subunit isoform expression of the Na,K-ATPase during postnatal development of the rat.

No description available.

No description available.

Murine decidual cells were examined for surface markers common to lymphomyeloid cells and for unique lineage specific markers by radioautography. They were shown to be Thy-1('+), Mac-1('(+OR-)), I-A('-), I-J('-) and Lyt('-). Two protein-A binding, IgG2b isotype monoclonal antibodies recognizing unique decidual cell surface antigen(s) were produced by immunizing virgin CBA mice with syngeneic decidual cells. Using radioautography and immunofluorescence, the incidence of decidual cells expressing these antigen(s) was found to rise with advancing gestation. They were also present on decidual cells of other mouse strains, rats, and humans, but not detectable on lymphomyeloid cells in any of these species. / Leukocyte subsets infiltrating the murine decidua basalis were also characterized and quantitated. A biphasic rise in total cellularity, more pronounced during allogeneic than syngeneic pregnancy, was attributable primarily to decidual cells and to a smaller extent lymphocytes, amongst which null (IgM('-), Thy-1('-)) cells predominated. Lyt-1 cells were the most frequent T cell subset, however, a terminal rise in the Lyt-2 subpopulation occurred in allogeneic pregnancy. Only a subpopulation of macrophages expressed I-A and all expressed Mac-1 antigens. / These findings provide a basis for further functional studies of the decidua.

Introduction: One of the many sources of low back pain (LBP) is the degeneration of intervertebral discs (IVDs), a condition called degenerative disc disease (DDD). Degeneration of IVDs can occur as result of aging, trauma, or genetic predisposition. Better insight into the complex histological profile in healthy and degenerated IVDs will contribute to our understanding of the interplay between the degenerative processes and chronic pain. The knowledge gained will ultimately allow for the development of better diagnostic and therapeutic strategies towards the management of LBP. Standard hematoxylin–eosin (H&E) staining, alone or in combination with other methods, has been traditionally used to assess the normal biology and degeneration of IVDs. A novel technique has recently been developed that provides additional information than previous methods; specifically, a clear differentiation between various sub-regions of the disc. In this method, termed FAST staining, the samples are exposed to a combination of dyes, starting with Alcian blue that blocks the acidic glycoproteins of the tissue. This is followed by a Safranin-O staining through which all the rest of the neutral proteoglycans will be stained. Finally Tartrazine and Fast green are used in order to stain the remaining mucin and collagen moieties. FAST and H&E provide complementary information regarding the compartmental organization and matrix composition of the discs in different conditions of health and degeneration.The principal objective of the current study is to characterize the histological profile of healthy and degenerated human IVDs with a combination of H&E and FAST staining. We will also present some preliminary data regarding the study of innervation in these samples using fluorescent immunohistochemistry (IHC). This will allow future investigations on the relationship between pain and the innervation pattern. Methods: Tissue samples: Samples of degenerating IVDs were collected from chronic low back pain patients. Healthy control discs were obtained post-mortem from donors. Histology: FAST and H&E were performed on cryostat sections obtained from fixed, frozen disc samples. Assessment of innervation: The analysis of the innervation and vascularization in different parts of the discs was done through immunohistochemical mapping studies using specific markers for nerve fibers (anti-PGP 9.5 and anti- CGRP) and blood vessels (anti-PECAM 1). Results: FAST staining enables differentiation of the three regions of the disc: inner annulus fibrosus, outer annulus and nucleous pulposus. In addition, a promising protocol for IHC was adapted to this type of tissue.Conclusion: FAST staining provides detailed information about disorganization of extracellular matrix, proteoglycan changes and calcification during the process of degeneration. H&E and FAST are complementary as one gives more information about the cellular content while the other better identifies the disc compartments, respectively. In addition, sparse sensory innervation can be observed in the very outer regions of healthy human IVDs using immunohistochemical methods. / Introduction : La dégénérescence des disques intervertébraux (DIVs) est une des sources connues de lombalgie. Cette condition, reconnue sous le nom de discopathie dégénérative, peut être conséquente du vieillissement, d'un traumatisme ou de prédispositions génétiques. Un meilleur aperçu du profil histologique de DIVs sains et dégénérés contribuera à approfondir notre compréhension des effets du processus dégénératif sur la douleur chronique. De plus, l'avancée des connaissances contribuera au développement de meilleurs diagnostics ainsi qu'à de meilleures stratégies thérapeutiques lors de la prise en charge de patients souffrant de lombalgie. La méthode standard de coloration tissulaire à base d'hématoxyline-éosine (H&E), utilisée seule ou en combinaison avec d'autres colorants, est couramment employée pour évaluer la biologie des DIVs sains ou dégénérés. Récemment, une nouvelle méthode a été développée afin de fournir de l'information additionnelle comme la différentiation définie entre les diverses sous-régions du disque. En utilisant la coloration FAST, les échantillons sont exposés à une combinaison d'agents colorants. Dans un premier temps, le bleu alcien bloque les glycoprotéines acides du tissu. Les protéoglycanes neutres restants sont ensuite colorés par la solution de Safranine-O. Finalement, les colorants Tartrazine et Fast green sont utilisés afin de colorer les fragments moléculaires de mucines et de collagènes. Les colorations FAST et H&E procurent de l'information complémentaire en ce qui concerne la composition de la matrice ainsi que l'organisation structurale des disques et ce, en présence ou non de dégénérescence tissulaire. L'objectif primaire de l'étude est de caractériser les profils histologiques de DIVs humains, sains ou dégénérés, à l'aide des colorations FAST et H&E. De plus, des données préliminaires d'une étude complémentaire sur l'innervation de ces mêmes échantillons observée par immunohistochimie (IHC) sont présentées à la toute fin. Ces données ouvrent la porte pour de futures recherches afin de comprendre la relation entre le patron d'innervation des DIVs et la douleur. Méthodes : Échantillons : Des échantillons tissulaires de DIVs dégénérés ont été recueillis chez des patients ayant de la douleur chronique lombaire. Des disques sains contrôles ont été obtenus de donneurs post-mortem. Histologie : Des coupes de tissus congelés et fixés ont été obtenues à l'aide d'un cryostat, puis colorées à l'aide des méthodes FAST et H&E. Évaluation de l'innervation : L'analyse de l'innervation et de la vascularisation des diverses régions des disques s'est effectuée par une étude de localisation immunohistochimique utilisant des marqueurs spécifiques pour les fibres nerveuses (anti-PGP 9.5 et anti-CGRP) et les vaisseaux sanguins (anti-PECAM 1). Résultats : La coloration FAST permet la différentiation des trois régions principales du disque : l'anneau fibreux interne, l'anneau fibreux externe et le noyau pulpeux. Un protocole d'IHC efficace a été adapté spécifiquement pour ce tissu. Conclusion : La coloration FAST fournit de l'information détaillée sur la désorganisation de la matrice extracellulaire, les changements au niveau des protéoglycanes ainsi que sur la calcification discale survenant lors du processus de dégénérescence. Les colorations H&E et FAST sont complémentaires puisque, de façon respective, une donne de l'information sur le contenu cellulaire alors que l'autre identifie clairement les sous-régions composant le disque. À l'aide de méthodes d'immunohistochimie, il est possible d'identifier une innervation sensorielle dispersée dans les régions les plus externes de disques humains sains.

Four aspects of the function of the vas deferens were examined by light microscope immunocytochemistry of Bouin-fixed, paraffin-embedded material. First, the expression of aquaporin-1 by endothelial cells of the vascular channels in the lamina propria of the distal region, suggested water transport from the lumen of the vas deferens via the dilated intercellular spaces to underlying vascular channels, the function of which may be to concentrate sperm. Second, the expression of 3beta-hydroxysteroid dehydrogenase by principal cells was indicative of steroid synthesis. Third, the ability of the vas deferens to create a special environment for protecting spermatozoa in the lumen was investigated using anti-glutathione S-transferase (GST) antibodies. Both principal and basal cells showed varying degrees of GST expression in the different regions of the cauda and vas deferens, suggesting a complex, changing environment of substrates to which epithelial cells and sperm are subjected. Fourth, the epithelial cells lining the cauda epididymidis and vas deferens were investigated for their secretory and endocytic functions. (Abstract shortened by UMI.)

The perinuclear theca is a unique cytoskeletal element that encapsulates the nucleus of mammalian spermatozoa except for a narrow zone around the insertion of the sperm tail. It is composed of two distinct regions, a subacrosomal layer or perforatorium and, continuing caudally beyond the acrosomic system, the postacrosomal sheath. Gel electrophoresis revealed that the extracts were composed of several polypeptide bands, of which the 15.5, 25, 28, 32, 36, and 60 kDa bands were most prominent in the bull. Antibodies against the 15.5 and 60 polypeptides of the bull reacted monospecifically with their respective bands on immunoblots and immunogold labeled the perforatorium and the entire perinuclear theca, respectively. Antibodies against the 25, 28, 32, and 36 kDa polypeptides of the bull showed distinctive patterns of cross reactivity with other polypeptides on Western blots, but immunogold labeled exclusively the entire perinuclear theca. Similar manipulation of human spermatozoa revealed that this element was composed of three major polypeptides, including the 14, 15, and 67 kDa bands. The anti-15.5 (bull) antibody, in addition to an antibody against the 15 kDa protein of rat spermatozoa (raised in a previous study) each cross-reacted with the 15 and 67 kDa polypeptides on Western blots of the human perinuclear theca. Conversely, the anti-60 (bull) antibody and an anti-60 (rat) antibody each reacted monospecifically with the 67 kDa polypeptide of the human perinuclear theca. As well, the anti-60 (bull) antibody immunogold labeled the entire perinuclear theca of human spermatozoa. (Abstract shortened by UMI.)