Our recent study demonstrated that Notch pathway in Sertoli cells is involved in the regulation of blood-testis barrier proteins, claudins, in androgen-dependent manner. 0.001) in Sertoli cells. Knockdown of EVP-6124 (Encenicline) AR or ZIP9 as well as antiandrogen exposure experiments revealed that (i) action of androgens via both AR and ZIP9 controls expression, (iii) ZIP9-dependent pathway regulates expression. Our findings indicate a crosstalk between EVP-6124 (Encenicline) androgen and Notch signaling in Sertoli cells and point to cooperation of classical and non-classical androgen signaling pathways in controlling Sertoli cell function. expression in Sertoli cells [17,18]. Disruption of Notch signaling in adult testis results in increased apoptosis and spermatogenesis defects . In contrast to many signaling pathways, Notch pathway lacks intermediate actions and does not exploit downstream secondary messengers for signal amplification. Binding of a ligand to Notch receptor results in receptor cleavage and translocation of Notch intracellular domain name (NICD) to the nucleus, where it interacts with a transcription factor recombination signal binding protein (RBP-J), and Mastermind-like 1 . The Notch co-activator complex induces the expression of target genes belonging to hairy/enhancer of split (Hes) and Hes-related with YRPW (Tyr-Arg-Pro-Trp) EVP-6124 (Encenicline) motif 1 (Hey) families . Despite its simplicity, outcomes of the pathway activation may be diverse even in the same cell type due to its complex regulation at different levels [22,23]. Studies using various tissues and cellular models demonstrated that this pathway may also crosstalk with other signaling pathways and such interactions may determine the final outcome [24,25,26,27]. Androgens were identified as factors modulating activity of Notch signaling EVP-6124 (Encenicline) in several cell types. Long-term testosterone enanthate treatment increased the expression of activated Notch1 in muscle satellite cells of older men consistent with increased satellite cell replication . The importance for androgen-Notch conversation for the course of prostate development and morphogenesis was also exhibited [29,30]. DeFalco et al.  found that in mice testosterone plays a role in maintaining the balance between progenitor cells and differentiated fetal Leydig cells, regulating levels of JAG1 and Notch2. In respect to androgen action in Sertoli cells, particular attention was devoted to genes related to the development of the blood-testis barrier . Our recent study exhibited that Notch pathway in Sertoli cells is usually involved in the regulation of blood-testis barrier proteins, claudins, in androgen-dependent manner. Using RNAi technique to reduce the expression of androgen receptors, we provided evidence that the effects of Notch signaling on claudin-5 and claudin-11 are dependent on the activation of ZIP9 or AR, respectively . Notably, this was the first report on ZIP9 regulation by the Notch pathway. We have also found that reduced androgen production or signaling alters the expression of Notch receptors, ligands and effector genes in testes of pubertal rats . To provide deeper insight in androgen-Notch crosstalk in mammalian testis and reveal the mechanisms involved in this conversation, we aimed to explore the role of nuclear and membrane androgen receptors in the regulation of Notch pathway activation in rodent Sertoli cells in vitro. 2. Results Firstly, to investigate whether Notch pathway activity in Sertoli cells is usually directly regulated by androgens, relative level of expression, activated form of Notch1 receptor (N1ICD) and the expression of the target genes and were decided in two murine Sertoli cell lines TM4 (from 11C13 day-old mice; expressing both the AR and ZIP9) and 15P-1 (from 6 month-old mice; lacking the AR, but expressing ZIP9). We found that testosterone, in the range of serum and testicular concentrations 10?8 MC10?6 M [34,35,36], increased mRNA and protein expression of 0.05, 0.01, 0.001), ( 0.05, 0.01, 0.001) and ( 0.05, 0.001) in both cell lines (Figure 1aCd). The lowest concentration capable of effectively activating Notch pathway (10?8 M) was used in further experiments. Open in a separate window Physique 1 The effect of testosterone on and mRNA, and Notch1 intracellular domain name (N1ICD), HEY1 and HES1 protein expression in TM4 and 15P-1 Sertoli cell lines. Cells were treated with a vehicle (control, C), 10?8 M, 10?7 M or 10?6 M testosterone (T) for 24 h (a,b) Relative expression of mRNAs (RQ) was determined using quantitative real-time RT-PCR analysis. The expression values of the individual genes were EVP-6124 (Encenicline) normalized to the mean expression of and 0.05, ** 0.01, and *** 0.001. Next, to determine the involvement of AR and ZIP9 in the regulation of Notch pathway activity we knocked down each of the receptors using RNAi technique. In TM4 cells, knockdown of either AR or ZIP9 partly abrogated the effect of testosterone on RBP-J activity Sstr3 (Physique 2a) and 0.05, 0.01, 0.001), which suggests.