The Regulation of Sertoli Cell Micro-RNAs by TGFB3.

Abstract

Most events of spermatogenesis, including meiosis, spermiogenesis and spermiation, take place in a unique adluminal microenvironment created by the blood-testis barrier, which is in part comprised of specialised junctions between Sertoli cells. In the rat testis, restructuring of the blood-testis barrier occurs at stage VIII of the seminiferous epithelial cycle, to allow the movement of preleptotene spermatocytes from the basal to the adluminal compartment to prepare for meiosis. However, the molecular mechanism(s) by which this blood-testis barrier restructuring occurs are poorly understood. It is well known that members of the TGF beta superfamily, including TGFβ2, TGFβ3 and GDF9, are present at stage VIII and can disrupt blood-testis barrier integrity in vivo and in vitro. In other epithelial cell types (e.g. breast, kidney), TGFβ-induced cell junction restructuring is mediated, in part, via micro-RNAs (miRNAs), which are small non-coding RNAs that post-transcriptionally regulate gene expression. We therefore hypothesised that TGFβ3 can regulate miRNAs in Sertoli cells, which in turn control spermatogenesis by targeting junction-associated proteins important in the function of the blood-testis barrier. The aim of this study was to identify TGFβ3-regulated miRNAs in rat Sertoli cells in vitro, and to correlate these with potential junctional targets. Addition of TGFβ3 to rat Sertoli cells in primary culture caused both dose- and time-dependent decreases in Sertoli cell tight junction function; these decreases could be completely ablated by addition of a prodomain antagonist which specifically binds TGFβ3. Comparative microarray analysis was then used to characterise TGFβ3-specific miRNAs produced by rat Sertoli cells. Of a total of 353 miRNAs which were commonly expressed in both TGFβ3- and TGFβ3-antagonist treated cells, 13 were up-regulated by TGFβ3 >5-fold and 12 miRNAs were down- regulated >5-fold. Several of the up-regulated miRNAs (miR-615-3p, miR-139-3p, miR-349) were also present in high concentration in Sertoli cells. Bioinformatic prediction of miRNA-mediated pathways (DIANA LAB micro-RNA pathway analysis software) using the up-regulated miRNAs as a combined group revealed adherens junctions, focal adhesions, and the actin cytoskeleton, to be amongst the four top pathways represented. Each of these pathways has known roles in the specialised junctions between Sertoli cells which contribute to the blood-testis barrier, suggesting that TGFβ-mediated miRNAs may impact on their function. This is supported by recent evidence from our lab which demonstrated that FSH- and androgen-mediated miRNAs in Sertoli cells target cell adhesion pathways in a coordinated manner. We conclude that TGFβ3 mediates a discrete subset of Sertoli cell miRNAs which potentially impact the regulation of Sertoli cell junctions important in the function of the blood-testis barrier. Research supported by the National Health and Medical Research Council (Australia) Program Grant #494802 and Project Grant #1009144. (poster)