The Role of ZIP9 and Androgen Receptor in the Establishment of Tight Junctions between Adult Rat Sertoli Cells

SIMPLE SUMMARY: Haploid male germ cells that originate at the onset of puberty from diploid precursor cells would be attacked by the innate immune system if they were not protected by the blood–testis barrier. This dynamic structure consists of tight junction proteins formed between epithelial Sertoli cells of the seminiferous tubules. Testosterone is the main regulator of this structure. We establish here, however, that testosterone actions affecting tight junctions, contrary to a decades-old assumption, are not mediated through the classical androgen receptor found in the cytosol or nuclei but through membrane-bound ZIP9, a multifunctional protein that is both a zinc transporter and androgen receptor. Our investigation establishes that ZIP9 plays a significant role in the regulation of tight junction protein expression and tight junction formation between Sertoli cells and that the involvement of ZIP9 should be considered in future studies dealing with the maintenance and protection of male fertility. ABSTRACT: The blood–testis barrier (BTB) is formed from tight junctions (TJs) between Sertoli cells. This dynamic structure, which establishes an immune-privileged environment protecting haploid germ cells formed in puberty from cells of the innate immune system, protects male fertility. Testosterone produced in Leydig cells is one of the main regulators of TJ protein expression and BTB dynamics. Nevertheless, although it has been assumed that testosterone effects on TJs and BTB are mediated through the classical androgen receptor (AR), newer results call the importance of this receptor into question. ZIP9, a recently identified androgen receptor of plasma membranes, mediates testosterone effects that promote the expression of TJ proteins and TJ formation in a rat Sertoli cell line that lacks the classical AR. Although these findings suggest that ZIP9 mediates these testosterone effects, participation of the classical AR in these events cannot be excluded. Here we used immortalized adult rat Sertoli cells that express both ZIP9 and AR and addressed the involvement of these receptors in the stimulation of TJ protein expression and TJ formation in response to testosterone and to the androgenic peptide IAPG that acts via ZIP9. We find that both testosterone and IAPG trigger the so-called non-classical signaling pathway of testosterone and stimulate the expression of TJ-associated proteins and TJ formation. Silencing classical AR expression had no effect on the responses, whereas silencing of ZIP9 expression completely blocked them. Our results demonstrate that ZIP9 is the sole androgen receptor involved in the regulation of TJ protein expression and TJ formation at the BTB.