Supplementary Materialsjcm-09-00961-s001. peritubular area of the seminiferous tubules. The cells express easy Pidotimod muscle mass actin (ACTA2), calponin (CNN1), and other common cytoskeletal markers, as well as a quantity of genes for specific extracellular matrix proteins [10]. Accordingly, for example, the extracellular matrix of the peritubular cell wall contains, among others, collagens and elastin (ELN; observe images in the Human Protein Atlas; [11]). Due to their easy muscle-like phenotype, they are able to contract and unwind and thereby transport sperm [6,12]. Based on immunohistochemical studies of easy muscle mass markers, this important feature is usually reported Pidotimod to be reduced, or even lost in male (idiopathic) infertility [13]. Also, the easy muscle-like characteristics of peritubular cells develop only at puberty in primates [14], highlighting the Pidotimod importance for male fertility and implicating hormones in its regulation. In the present study, we analyzed the expression of GR in the testes of man and in a non-human primate species. We also employed HTPCs as a cellular model system to explore effects of GR activation in particular after application of dexamethasone (Dex), a frequently used synthetic glucocorticoid. We focused on the easy muscle mass and extracellular markers and show that Dex regulates easy muscle characteristics of HTPCs. 2. Experimental Section 2.1. Human and Monkey Samples Testicular biopsies for HTPC isolation and for immunohistochemistry were obtained from men 36C55 years of age (in total = 11) with obstructive azoospermia but normal spermatogenesis as explained [5,9,13]. The study was approved by the local Ethics Committee (Technical University or college of Munich, Faculty of Medicine; project 491/18S-KK), and scientific use of the cells was permitted by written educated consent from all the patients. The experiments were carried out in accordance with the relevant recommendations and regulations, and participants offered written consent. Post-mortem testicular samples were from rhesus monkeys (= 3) for 24 h using Rabbit Polyclonal to ENDOGL1 the firefly luciferase assay, CellTiter-Glo? Assay-kit (Promega, Mannheim, Germany) following a manufacturers protocol. The luminescence was measured inside a luminometer (BMG Labtech, Ortenberg, Germany). 2.4. Isolation of RNA and Protein Isolation of RNA and proteins from HTPCs of a total of 3C8 different donors was performed as previously explained [8,18]. 2.5. Reverse Transcription (RT-PCR) and Quantitative Real Time PCR (qPCR) Complementary DNA (cDNA) was synthesized from 0.4 g RNA using dN12 random primer followed by conventional PCR as described earlier [8]. qPCR measurements using the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany) were conducted within the LightCycler 96? System (Roche Diagnostics, Penzberg, Germany) as layed out before [8]. Samples were run in duplicate and analyzed using a CT calculation method [19]. Primers (observe Table S1) were designed by the 3web software spanning at least one intron when possible. Amplicons were verified by agarose gel electrophoresis with Midori Green Advance DNA stain (Nippon Genetics Europe, Dren, Pidotimod Germany) followed by sequence analysis (GATC, Konstanz, Germany). 2.6. Western Blotting Western blot analysis was performed with HTPCs whole cell lysates as explained [20]. Protein samples (10C15 g) were separated via 10% SDS-PAGE and subjected to immunoblotting. Membranes were incubated overnight with the same anti-GR antibody (1:2500) as utilized for immunohistochemistry at 4 C, followed by IRDye800 labeled secondary antibodies for 1 h. Membranes were scanned with the infrared-based Odyssey Imaging System (Li-Cor, Bad Pidotimod Homburg, Germany) and quantified using the Image Studio software. Results were normalized to -actin (1:5000, anti–actin mouse monoclonal antibody A5441, Sigma-Aldrich, St. Louis, MO, USA) providing like a loading control. 2.7. Immunohistochemistry (IHC) and Immunocytochemistry (ICC) Paraffin-embedded samples from individuals with normal spermatogenesis were processed according to the avidin-biotin-peroxidase method [8]. HTPCs in tradition were fixed with chilly 4% paraformaldehyde for 15 min, and IF staining was performed having a fluorescence-tagged supplementary antibody (1:800; goat -rabbit Alexa-488, Lifestyle technology, Carlsbad, CA, USA) as defined with slight adjustments [21]. For both methods, an anti-NR3C1 (=GR) principal antibody (1:500 for IHC, 1:50 for ICC; affinity-purified, polyclonal rabbit anti-human NR3C1, HPA004248 Sigma Prestige Antibodies, St. Louis, MO, USA) was utilized, which identifies both GR and GR isoforms. In further IHC tests, the following principal antibodies had been utilized: anti-ACTA2 (1:200, mouse monoclonal anti-actin, Sigma A5228, St. Louis, MO, USA), Col1 (1:200, affinity-purified, polyclonal rabbit anti-Col1, R1038, Origene, Rockville, MD,.