Abstract
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.
Highlights
Sertoli cells (SCs) are fundamental for studies of the blood testis barrier (BTB) and testicular immune privilege, as well as the analysis of spermatogenesis-associated events in vitro and in vivo [1]
We describe a new protocol for the isolation and long-term maintenance without the need of repeated animal sacrifice of adult rat SCs, which is based on conditional reprogramming
Complete conditioned medium (CM) supported presumable SC clusters to reach near confluency (Figure 2F)
Summary
Sertoli cells (SCs) are fundamental for studies of the blood testis barrier (BTB) and testicular immune privilege, as well as the analysis of spermatogenesis-associated events in vitro and in vivo [1]. Various methods have been developed for primary SC isolation, such as enzymatic digestion [2], sometimes followed by fluorescence-activated cell sorting (FACS) [3,4] These methods have limitations such as the toxicity of fluorochromes (e.g., propidium iodide) [2] and the possible harmful effects of the laser beam used in FACS [5]. For enzymatic digestion, a high number of animals is needed and the harsh enzymatic treatment results often in low yields and a damaged cell surface [2,6,7,8,9] All of these protocols for the isolation of primary SCs have similar problems such as repeated sacrifice of animals, lack of purity, a decline in hormone responsiveness after few passages, and most importantly, the cells have a limited life span in culture
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