Notch signaling regulates nuclear androgen receptor AR and membrane androgen receptor ZIP9 in mouse Sertoli cells

Background: Notch signaling pathway is involved in contact-dependent communication between the cells of seminiferous epithelium, and its proper activity is important for undisturbed spermatogenesis.

Objectives: The aim was to assess the effect of Notch pathway inhibition on the expression of nuclear (AR) and membrane (ZIP9) androgen receptors and androgen-regulated genes, claudin-5 and claudin-11, in TM4 mouse Sertoli cell line.

Materials and methods: DAPT (γ-secretase inhibitor) treatment and recombination signal binding protein silencing were employed to reduce Notch signaling, whereas immobilized ligands were used to activate Notch pathway in TM4 cells. To reveal specific effect of each androgen receptor, AR or ZIP9 silencing was performed.

Results: Notch pathway inhibition increased the expression of AR and ZIP9 mRNA and proteins (p < 0.01; p < 0.05) in TM4 cells, whereas incubation with Notch ligands, rDLL1 or rJAG1, reduced AR (p < 0.01; p < 0.001) and ZIP9 (p < 0.05; p < 0.01) expressions, respectively. Testosterone enhanced the expression of both receptors (p < 0.05; p < 0.01). Androgen-regulated claudin-5 and claudin-11 (p < 0.01; p < 0.001) and cAMP (p < 0.001) were elevated in Notch-inhibited cells, while activation of Notch signaling by DLL1 or JAG1 reduced claudin-11 or claudin-5 level (p < 0.01; p < 0.001), respectively.

Discussion: Our findings indicate opposite effect of Notch and androgen signaling on the expression of androgen receptors in TM4 cells. We demonstrated that AR expression is regulated by DLL1-mediated Notch signaling, whereas JAG1 is involved in the regulation of ZIP9. The expression of both claudins and cAMP production is under inhibitory influence of Notch pathway. The effects of Notch signaling on claudin-5 and claudin-11 expression are mediated by ZIP9 and AR, respectively.

Conclusion: Notch signaling may be considered as an important pathway controlling Sertoli cell physiology, and its alterations may contribute to disturbed response of Sertoli cells to androgens.