Cyclical and Patch-Like GDNF Distribution along the Basal Surface of Sertoli Cells in Mouse and Hamster Testes

BACKGROUND AND AIMS: In mammalian spermatogenesis, glial cell line-derived neurotrophic factor (GDNF) is one of the major Sertoli cell-derived factors which regulates the maintenance of undifferentiated spermatogonia including spermatogonial stem cells (SSCs) through GDNF family receptor α1 (GFRα1). It remains unclear as to when, where and how GDNF molecules are produced and exposed to the GFRα1-positive spermatogonia in vivo. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show the cyclical and patch-like distribution of immunoreactive GDNF-positive signals and their close co-localization with a subpopulation of GFRα1-positive spermatogonia along the basal surface of Sertoli cells in mice and hamsters. Anti-GDNF section immunostaining revealed that GDNF-positive signals are mainly cytoplasmic and observed specifically in the Sertoli cells in a species-specific as well as a seminiferous cycle- and spermatogenic activity-dependent manner. In contrast to the ubiquitous GDNF signals in mouse testes, high levels of its signals were cyclically observed in hamster testes prior to spermiation. Whole-mount anti-GDNF staining of the seminiferous tubules successfully visualized the cyclical and patch-like extracellular distribution of GDNF-positive granular deposits along the basal surface of Sertoli cells in both species. Double-staining of GDNF and GFRα1 demonstrated the close co-localization of GDNF deposits and a subpopulation of GFRα1-positive spermatogonia. In both species, GFRα1-positive cells showed a slender bipolar shape as well as a tendency for increased cell numbers in the GDNF-enriched area, as compared with those in the GDNF-low/negative area of the seminiferous tubules. CONCLUSION/SIGNIFICANCE: Our data provide direct evidence of regionally defined patch-like GDNF-positive signal site in which GFRα1-positive spermatogonia possibly interact with GDNF in the basal compartment of the seminiferous tubules

Deficiency in interleukin-18 promotes differentiation of brown adipose tissue resulting in fat accumulation despite dyslipidemia

Abstract Background The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis. Methods Il18 −/− male mice were generated on the C57Bl/6 background, and littermate C57Bl/6 Il18 +/+ male mice were used as controls. To reveal the direct effect of IL-18, primary cell cultures derived from both mice were established. Moreover, for molecular analysis, microarray, quantitative reverse transcription PCR and western blotting were performed using 6 and 12 weeks old mice. To evaluate the short- and long-term effects of IL-18 on BAT, recombinant IL-18 was administered for 2 and 12 weeks, respectively. Results Compared with Il18 +/+ mice, BAT of Il18 −/− mice showed earlier differentiation and lipid accumulation. To examine the direct effect of IL-18 on BAT, BA cell cultures were established. Myogenic factor 5-expressing adipose precursor cells were extracted from Il18 +/+ and Il18 −/− mice. PR domain containing 16 (PRDM16), a differentiation inducer, was strongly expressed in Il18 −/− BAs, and uncoupling protein 1, a thermogenic and differentiation marker, was upregulated, resulting in the promotion of BA differentiation. Moreover, PRDM16-dependent and independent molecules related to BAT function, such as fibroblast growth factor 21, were activated. These findings were confirmed by comparing Il18 +/+ and Il18 −/− mice at 6 and 12 weeks of age. Additional analyses of the molecular mechanisms influencing the ‘Quantity of adipocytes’ identified three associated genes, apolipoprotein C3 (Apoc3), insulin-induced gene 1 (Insig1) and vitamin D (1,25-dihydroxyvitamin D3) receptor (Vdr). Intravenous administration of IL-18 not only significantly improved the expression of some of these genes, but it also significantly decreased the adipocytes’ size. Conclusions This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance

Additional file 2: Figure S1. of Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke

Localization and characterization of Iba1+ microglia after ischemic stroke. Immunohistochemical localization of Iba1 using the DAB reaction (A–L). In sham-operated mice, resting Iba1+ microglia showing a ramified morphology were observed in the MCA areas of the cortex (A–C). On post-ischemia day 3, many Iba1+ microglia were observed in peri-ischemic areas, with some cells exhibiting an ameboid-like, activated microglial morphology (D–F). On days 5 (G–I) and 7 after ischemia (J–L), most of the Iba1+ microglia exhibited an ameboid morphology and were localized in and around the ischemic areas. The numbers of Iba1+ cells in the ischemic core and peri-ischemic areas are indicated (M). The populations of ramified- or ameboid-like Iba1+ microglia in the ischemic core and peri-ischemic areas are shown (N). Scale bars = 100 μm (B, E, H, K) and 50 μm (C, F, I, L). Abbreviations: DAB, diaminobenzidine; Iba1, ionized calcium binding adaptor molecule 1; MCA, middle cerebral artery. (PDF 543kb

Additional file 1: Supplemental information. of Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke

Methods for Additional files (2-4) are available in Supplemental methods of this file. (DOCX 56.8kb

Additional file 4: Figure S3. of Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke

Localization and characterization of CD68+ cells following ischemia. CD68+ cells were rarely observed at the ischemic core and peri-ischemic areas on post-stroke day 3 (A–D) (Iba1 (B, C: red), CD68 (B, D: green), DAPI (B–D: blue)). Although only a small number of CD68+ cells were observed in these areas, on post-stroke day 5 (E–H) (Iba1 (F, G: red), CD68 (F, H: green), DAPI (F–H: blue)) and 7 (I–L) (Iba1 (J, K: red), CD68 (J, L: green), DAPI (J–L: blue)), some of them expressed Iba1 (arrows). However, these CD68+ cells rarely expressed PDGFRβ at post-stroke day 5 (M–P) (PDGFRβ (N, O: red), CD68 (N, P: green), DAPI (N–P: blue)) or day 7 (Q–T) (PDGFRβ (R, S: red), CD68 (R, T: green), DAPI (R–T: blue)). Scale bars = 20 μm (B, F, J, N, R). Abbreviations: Iba1, ionized calcium binding adaptor molecule 1; PDGFRβ, platelet-derived growth factor receptor-β. (PDF 398kb

Additional file 3: Figure S2. of Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke

PDGFRβ+ iPCs express the microglial marker CD206. On post-stroke day 3, some PDGFRβ+ cells within ischemic areas represent the microglial marker CD206 (A–D) (PDGFRβ (B, C: red), CD206 (B, D: green), DAPI (B–D: blue)) (arrows). Scale bars = 20 μm (B). Abbreviations: PDGFRβ, platelet-derived growth factor receptor-β. (PDF 209kb