A protocol for the isolation and long-term propagation of adult rat Sertoli cells (SCs) using conditional reprogramming (CR) was developed and the formation of tight junctions as an in vitro model for the blood testis barrier (BTB) was studied. Three pure primary SC lines were isolated successfully and maintained for several months without significant changes in expression levels of SC-typical markers such as SRY-box transcription factor 9 (SOX9), transferrin, clusterin, androgen receptor (AR), and GATA binding protein 1 (GATA1). In addition to AR expression, the tight junction proteins, zonula occludens-1 (ZO-1) and the junctional adhesion molecule-3 (JAM-3), were upregulated and the SC barrier integrity was enhanced by testosterone. Peritubular/myoid cells did not increase the tightness of the SC. The cytokines, interleukin-6 (IL-6), bone morphogenetic protein-2 (BMP2), and transforming growth factor beta-3 (TGF-β3), negatively affected the tightness of the SC barrier. We have established a protocol for the isolation and long-term propagation of highly pure primary adult rat SCs, which are able to respond to androgen treatments, to form tight junctions and to maintain the mRNA expression of SC-specific genes. By applying this new method, adult SCs can now be analyzed in more detail and might serve as an in vitro model for the study of many SC functions.
Keywords: Sertoli cells; androgen receptor; blood testis barrier; conditional reprogramming; tight junctions.