Sensitivity of spermatogonia to irradiation varies with age in pre-pubertal ram lambs

Although germ cells from donor rams transplanted into irradiated recipient testes have produced donor derived offspring, efficiency is low. Further optimization of recipient irradiation protocols will add precision to the depletion of recipient spermatogonia prior to germ cell transplant. Three irradiation doses (9,12,15 Gy) were administered to ram lambs aged 14 weeks (Group 1) and 20 weeks (Group 2), then testicular biopsies were collected 1, 2 and 3 months after irradiation. At 1 month after irradiation of Group 1, only the largest dose (15 Gy) reduced spermatogonia numbers below 10% of non-irradiated controls, whereas in Group 2 lambs, each irradiation dose reduced spermatogonia below 10% of controls. In both Groups, fewer differentiated germ cells were present in seminiferous tubules compared to controls. At 2 months after irradiation, spermatogonia numbers in both Groups increased more than sixfold to be similar to controls, whereas fewer differentiated germ cells were present in the tubules of both Groups. At 3 months in Group 1, each irradiation dose reduced spermatogonia numbers t

Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity

INTRODUCTION: The regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface. METHODS: We have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding. RESULTS: Cell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells. CONCLUSION: We show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential

The gastrointestinal nematode Trichostrongylus colubriformis down-regulates immune gene expression in migratory cells in afferent lymph

Background: Gastrointestinal nematode (GIN) infections are the predominant cause of economic losses in sheep. Infections are controlled almost exclusively by the use of anthelmintics which has lead to the selection of drug resistant nematode strains. An alternative control approach would be the induction of protective immunity to these parasites. This study exploits an ovine microarray biased towards immune genes, an artificially induced immunity model and the use of pseudo-afferent lymphatic cannulation to sample immune cells draining from the intestine, to investigate possible mechanisms involved in the development of immunity.\ud \ud Results: During the development of immunity to, and a subsequent challenge infection with Trichostrongylus colubriformis, the transcript levels of 2603 genes of cells trafficking in afferent intestinal lymph were significantly modulated (P < 0.05). Of these, 188 genes were modulated more than 1.3-fold and involved in immune function. Overall, there was a clear trend for down-regulation of many genes involved in immune functions including antigen presentation, caveolar-mediated endocytosis and protein ubiquitination. The transcript levels of TNF receptor associated factor 5 (TRAF5), hemopexin (HPX), cysteine dioxygenase (CDO1), the major histocompatability complex Class II protein (HLA-DMA), interleukin-18 binding protein (IL-18BP), ephrin A1 (EFNA1) and selenoprotein S (SELS) were modulated to the greatest degree.\ud \ud Conclusions: This report describes gene expression profiles of afferent lymph cells in sheep developing immunity to nematode infection. Results presented show a global down-regulation of the expression of immune genes which may be reflective of the natural temporal response to nematode infections in livestock

Systems biology of ovine intestinal parasite resistance: disease gene modules and biomarkers

This study reports on the molecular systems biology of gastrointestinal nematode (GIN) infection and potential biomarkers for GIN resistance in sheep. Microarray gene expression data were obtained for 3 different tissues at 4 time points from sheep artificially challenged with two types of nematodes, Haemonchus contortus (HC) and Trichostrongylus colubriformis (TC). We employed an integrated systems biology approach, integrating 3 main methods: standard differential gene expression analyses, weighted gene co-expression network analyses (WGCNA) and quantitative genetic analyses of gene expression traits of key biomarkers. Using standard differential gene expression analyses we identified differentially expressed genes (DE) which responded differently in sheep challenged with HC compared to those challenged with TC. These interaction genes (e.g. MRPL51, SMEK2, CAT, MAPK1IP1 and SLC25A20A) were enriched in Wnt receptor signalling pathway (p = 0.0132) and positive regulation of NFκβ transcription factor activity (p = 0.00208). We report FCER1A, a gene encoding a high-affinity receptor for the Fc region of immunoglobulin E, which is linked to innate immunity to GIN in sheep. Using weighted gene co-expression network analysis (WGCNA) methods, we identified gene modules that were correlated with the length of infection (disease modules). Hub genes (with high intramodular connectivity) were filtered further to identify biomarkers that are related to the length of infection (e.g. CAT, FBX033, COL15A1, IGFBP7, FBLN1 and IgCgamma). The biomarkers we found in HC networks were significantly associated with functions such as T-cell and B-cell regulations, TNF-alpha, interleukin and cytokine production. In TC networks, biomarkers were significantly associated with functions such as protein catabolic process, heat shock protein binding, protein targeting and localization, cytokine receptor binding, TNF receptor binding, apoptosis and IGF binding. These results provide specific gene targets for therapeutic interventions and provide insights into GIN infections in sheep which may be used to infer the same in related host species. This is also the first study to apply the concept of estimating breeding values of animals to expression traits and reveals 11 heritable candidate biomarkers (0.05 to 0.92) that could be used in selection of animals for GIN resistance.Haja N. Kadarmideen, Nathan S. Watson-Haigh and Nicholas M. Andronico

Systems genetics analysis reveals gene modules and heritable biomarkers for sheep intestinal parasite resistance

Systems genetics methods were applied to microarray gene expression profiling data from a sheep gastrointestinal nematode (GIN) challenge experiment that was designed to detect genes associated with resistance to GIN. Analyses went beyond just detecting and annotating differentially expressed (DE) genes. This included detection of co-expressed (CE) gene modules associated with the duration of infection, essential hub genes, functional enrichment and pathway analyses. Results revealed that DE genes were highly enriched in functions such as cell-mediated and humoral immunological response to GIN. Further, heritabilities were estimated for expression phenotypes of such candidate biomarkers (range 0.05 to 0.9 with high s.e.) indicating their potential for expression-assisted selection. Hence, the systems genetics method is a key step in identifying biologically relevant and heritable genes/biomarkers amongst several sets of DE genes. This approach would provide specific targets for breeding and therapeutic interventions.H. N. Kadarmideen, N. Andronicos, and N. S. Watson-Haig