Tetrapeptides Modelled to the Androgen Binding Site of ZIP9 Stimulate Expression of Tight Junction Proteins and Tight Junction Formation in Sertoli Cells

SIMPLE SUMMARY: Androgens such as testosterone act either through the nuclear androgen receptor or through the plasma membrane-bound androgen receptor ZIP9. Androgens are crucial for male fertility. In the testis they stimulate the formation of tight junctions between Sertoli cells, thereby creating an immune-privileged environment for the development of fertile sperm known as the blood–testis barrier. Androgens are also absolutely required for spermatogenesis. Nevertheless, when low testosterone is supplemented by exogenous testosterone (e.g., in testosterone replacement therapy), it triggers a loss of sperm, and thus and infertility, by a negative feed-back mechanism based on the hypothalamic-pituitary-gonadal axis. In order to investigate this discrepancy, the effects of small peptides that fit within the androgen binding site of ZIP9 were tested for their effects on the expression of proteins involved in the formation of tight junctions. Our results provide evidence that all three peptides tested act through ZIP9 as androgens and promote tight junction protein expression and tight junction formation. They can therefore act as surrogates of testosterone for the formation and maintenance of the blood–testis barrier. ABSTRACT: Androgens stimulate the expression of tight junction (TJ) proteins and the formation of the blood–testis barrier (BTB). Interactions of testosterone with the zinc transporter ZIP9 stimulate the expression of TJ-forming proteins and promote TJ formation in Sertoli cells. In order to investigate androgenic effects mediated by ZIP9 but not by the nuclear androgen receptor (AR), the effects of three tetrapeptides fitting the androgen binding site of ZIP9 were compared with those induced by testosterone in a Sertoli cell line expressing ZIP9 but not the AR. Three tetrapeptides and testosterone displaced testosterone-BSA-FITC from the surface of 93RS2 cells and stimulated the non-classical testosterone signaling pathway that includes the activation of Erk1/2 kinases and transcription factors CREB and ATF-1. The expression of the TJ-associated proteins ZO-1 and claudin-5 was triggered as was the re-distribution of claudin-1 from the cytosol to the membrane and nucleus. Furthermore, TJ formation was stimulated, indicated by increased transepithelial electrical resistance. Silencing ZIP9 expression by siRNA prevented all of these responses. These results are consistent with an alternative pathway for testosterone action at the BTB that does not involve the nuclear AR and highlight the significant role of ZIP9 as a cell-surface androgen receptor that stimulates TJ formation.