Presence of N-acetylgalactosamine/galactose residues on bronchioloalveolar cells during rat postnatal development

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during post-natal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase-conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, at one-, four- and eight-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in one- and four-day-old animals and adult animals. These results were not so manifest in eight-day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in O- and N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.

A single simple procedure for dewaxing, hydration and heat-induced epitope retrieval (HIER) for immunohistochemistry in formalin fixed paraffin-embedded tissue

<p>Heat-induced epitope retrieval (HIER) is widely used for immunohistochemistry on formalin fixed paraffin-embedded tissue and includes temperatures well above the melting point of paraffin. We therefore tested whether traditional xylene-based removal of paraffin is required on sections from paraffin-embedded tissue, when HIER is performed by vigorous boiling in 10 mM Tris/0.5 mM EGTA-buffer (pH=9). Immunohistochemical results using HIER with or without prior dewaxing in xylene were evaluated using 7 primary antibodies targeting proteins located in the cytosol, intracellular vesicles and plasma membrane. No effect of omitting prior dewaxing was observed on staining pattern. Semiquantitative analysis did not show HIER to influence the intensity of labelling consistently. Consequently, quantification of immune labelling intensity using fluorescent secondary antibodies was performed at 5 dilutions of primary antibody with and without prior dewaxing in xylene. No effect of omitting prior dewaxing on signal intensity was detectable indicating similar immunoreactivity in dewaxed and non-dewaxed sections. The intensity of staining the nucleus with the DNA-stain ToPro3 was similarly unaffected by omission of dewaxing in xylene. In conclusion, the HIER procedure described and tested can be used as a single procedure enabling dewaxing, hydration and epitope retrieval for immunohistochemistry in formalin fixed paraffin-embedded tissue.</p>

270 PROGRESSIVE INCORPORATION OF CENEXIN IS RELATED TO SPERM MATURATION DURING EPIDIDYMAL TRANSIT IN THE DOMESTIC CAT

The sperm centrosome is an essential organelle playing a key role just after penetration into the oocyte. It serves to organise the sperm aster, which is required for syngamy and formation of the first mitotic spindle. It is also associated with acquisition of motility during epididymal transit. Previously, we demonstrated that testicular spermatozoa exhibit reduced developmental potential after oocyte injection due to the presence of an immature centrosome [Comizzoli et al. 2006 Biol. Reprod. 75, 252–260]. Centrosome and flagellum maturation naturally occur during epididymal transit where secreted proteins impart changes on the sperm to acquire its functional properties. The objective of this study was to better understand centrosome and flagellum maturation and identify key proteins that could be used to artificially mature testicular spermatozoa. Specifically, we focused our effort on cenexin, a protein that has been reported to aid in maturation of the flagellum and somatic cell centrosome. Epididymides were dissected from adult cat testes (>1 yr old). Spermatozoa were then extracted from the different regions (caput, corpus, cauda, and vas deferens) by slicing with a scalpel blade in phosphate buffered saline at 37°C and processed separately. Control samples were also collected from the rete testis. After recording sperm motility and forward progressive movement (FPM, from 0 = immotile to 5 = fast and straight), cells were fixed in 4% paraformaldehyde and immunostained with anti-cenexin antibodies labelled with a fluorescent probe. The proportion of cells with cenexin at the location of the centrosome and the intensity of immunofluorescence were quantified (n = 8 and 4 testes, respectively). The same methods were followed for detection of cenexin in the tail portion (n = 4 testes). Statistical analyses were conducted using repeated-measures and treatments were further compared using either a protected Tukey's or F-test for orthogonal contrasts. The proportion of sperm with cenexin localised at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis and highest proportion in the cauda (45 v. 81%, T28 = 4.65, P < 0.0001). Among the labelled sperm, the intensity of immunofluorescence also significantly increased from the testis to vas deferens (4.33 v. 8.57 mean grey value; T12 = 3.29, P < 0.0065). Both motility and FPM increased from the testis to cauda segment (0 v. 93%, F4,15 = 13.53, P < 0.0001 and 0 v. 3.8 FPM, F4,15 = 26.67, P < 0.0001); however, the proportion with cenexin in the tail (range, 20 to 36%) as well as the labelling intensity (range, 3.14 to 5.26 mean grey value) did not change (P > 0.05) along the tract. These results clearly indicate that cenexin may be associated with centrosome but not flagellum maturation. Epididymal epithelial cells and luminal fluid from each segment are being examined to better understand the source of cenexin secretion and its incorporation into spermatozoa. Results from these studies will aid in further understanding the physiology of sperm maturation during epididymal transit and increase male fertility preservation options.

NKX3.1 is expressed in ER-positive and AR-positive primary breast carcinomas

AimsNKX3.1 is an androgen-regulated tumour suppressor gene that is downregulated in prostate carcinoma. Immunohistochemistry for NKX3.1 is primarily specific for prostatic-derived tumours and tissue but is reported in a small number of breast carcinomas. NKX3.1 is also shown to inhibit estrogen receptor (ER) signalling in breast carcinoma models. Here, we investigate labelling of NKX3.1 in invasive ductal (IDC) and lobular (ILC) carcinomas of the breast with full characterisation of ER, progesterone receptor (PR), androgen receptor (AR) and Her2 status.MethodsTissue microarrays of 86 primary IDC and 37 ILC were labelled for NKX3.1. The IDC consisted of 20 luminal A, 7 luminal B, 14 Her2, and 45 triple negative carcinomas. The ILC consisted of 34 luminal A and 3 luminal B cases. NKX3.1 expression was scored as percentage nuclear labelling and labelling intensity.ResultsNuclear NKX3.1 labelling was seen in 2 IDC (2%) and 10 ILCs (27%). labelling intensity was weak in all cases (1–100% nuclear positivity). Positive NKX3.1 labelling was significantly associated with ILC (p<0.0001). NKX3.1 labelling was seen only in ER and AR-positive carcinomas, which showed a significant correlation (p=0.0003 and p=0.0079, respectively). Expression was not correlated with tumour stage, size, Her2 expression, presence of lymph node metastases or age.ConclusionsThis is the first study to evaluate NKX3.1 expression in breast carcinomas with known ER, PR, AR and Her2 status. Further studies are needed to evaluate what potential role NKX3.1 plays in ER and AR signalling and hormonal treatment response in breast carcinomas.

Genome reprogramming during the first cell cycle in in vitro produced porcine embryos

Conflicting data still exist regarding the extent of paternal pronuclear DNA demethylation in one cell-stage mammalian embryos. Demethylation of paternal pronuclear DNA was observed in <i>in vitro</i> produced porcine zygotes, whereas <i>in vitro</i> produced embryos do not show any or only weak paternal genome demethylation. In our experiments, we have used and compared two <i>in vitro</i> techniques commonly used for <i>in vitro</i > embryo production (<i>in vitro</i> fertilization and intracytoplasmic sperm injection) and then we evaluated the extent of labelling in both these groups after 5-methylcytosine (5-MeC) or dimethyl H3/K9 labelling. We have found no differences in the methylation pattern between both those techniques used for the production of embryos. Moreover, we did not detect any demethylation of paternal DNA after 5-MeC labelling at all. Contrary to this, labelling with dimethyl H3/K9 antibodies showed differences in labelling intensity between maternal and paternal genomes in 42% of zygotes after <i>in vitro</i> fertilization and in 44% of zygotes after intracytoplasmic sperm injection. Our results indicate that <i>in vitro</i> matured pig oocytes exhibit rather inconsistent methylation patterns. This inconsistency probably resulted from insufficient cytoplasmic maturation of oocytes and to a lesser extent from the <i>in vitro</i> technique for embryo production.

Octopamine-like immunoreactivity in the brain and suboesophageal ganglion of two parasitic wasps, Cotesia glomerata and Cotesia rubecula

Two closely related parasitoid wasp species, Cotesia glomerata L. and C. rubecula Marshall (Hymenoptera: Braconidae), differ in their display of associative learning and memory during host searching. As octopamine is involved in learning and memory in insects we investigated octopaminergic pathways in the brain and suboesophageal ganglion (SOG) of the two wasps. We used an anti-octopamine antibody and subsequent whole mount analysis using a confocal laserscanning microscope and pertinent software. Three groups of octopaminergic cells were located in the brain and suboesophageal ganglion. One group was located near the antennal lobes and consisted of six to eight cell bodies. A second group was located ventrally in the SOG and was most likely formed by ventral unpaired median (VUM) and VCBN (ventral cell body neurite) neurons. A third group was located in the pars intercerebralis and consisted of four to six cells. Octopamine-like immunoreactivity was furthermore present in the central body, protocerebral bridge, the SOG, antennal lobe, near the alpha and beta lobes of the mushroom bodies and in the mushroom body calyces. Due to the used methods and a high variability in staining intensity it was not possible to detect if there were any differences in the number of neurons, in arborisation patterns or in labelling intensity between the two wasp species.

Bovine abnormal preimplantation embryos: analysis of segregated cells occurring in the subzonal space and/or blastocoele cavity for their nuclear morphology and persistence of RNA synthesis

Bovine embryos in the early blastocyst/blastocyst stage were analysed by [5-3H]uridine labelling followed by electron microscopic autoradiography. In normal control embryos an intact zona pellucida, evenly developed blastomeres and a transparent perivitelline space were seen. In this group, the blastomeres of the trophoblast and embryoblast showed high homogeneous labelling localised in the nucleoplasm and even more intense labelling in the nucleolus. On the contrary, in addition to evident cytoplasmic disintegration, a clearly different labelling pattern and a low labelling intensity were observed in the nuclei of the segregated cells in the subzonal space and in those free in the blastocoele cavity. A typical nuclear morphological feature of these blastomeres was chromatin marginalisation, similar to that observed in embryos treated with actinomycin D for transcription inhibition. It is concluded that the segregated cells are arrested in their further differentiation.

Transcriptive and replicative activity of the X chromosome in an autosomal segmental hyperploid in Drosophila and its significance

In the present investigation the transcription and replication patterns have been examined in different segments of the X chromosome and in certain specific segments (88B-92A) of an autosomal segmental hyperploid in which an extra segment 88B-92A (3R) is translocated to the X chromosome in addition to the normal two doses. Transcriptive activity monitored by [3H]uridine-labelling of these autosomal hyperploids reveals an enhanced hyperactivity of the male X chromosome while the female X chromosomes show no change in their activity. [3H]thymidine autoradiograms reveal that while the labelling frequencies of most replicating sites are distinctly lowered in the autosomal hyperploid males, no change within sexes is resolvable with regard to labelling-intensity profile. Furthermore, the X-autosome labelling frequency relation shows a distinct deviation from linearity, suggesting multiple events that lead to a higher template form of the X chromosome. These findings lead us to suggest that the signals emanating from autosome(s) do not interfere with the primary modulation inherent in the X chromosome, but act on a modulated organization of the same at a second step evoking higher activity in the male X chromosome. The results further reveal that the gene activity of the X chromosome remains unaffected by the pattern of pairing of the autosomal segments.