Activin A is a pleiotropic factor that stimulates proliferation and inhibits differentiation of Sertoli cells during embryogenesis and early post-natal life. At puberty, serum activin levels decline markedly as Sertoli cells cease proliferating and differentiate in order to sustain adult spermatogenesis. We recently discovered that activin A can re-program differentiated rat Sertoli cells into a proliferative, functionally immature phenotype, characterised in vitro by a loss of intercellular tight junctions that form the blood-testis barrier, and the re-expression of immature protein markers. However, the molecular mechanism(s) by which activin exerts these actions remain unknown. We hypothesised that activin A targets key Sertoli cell functions and pathways, including regulation of micro-RNAs (miRNAs), which promote the de-differentiation of Sertoli cells to a functionally juvenile phenotype. We aimed to identify activin A-regulated miRNAs in post-pubertal (d20) rat Sertoli cells in vitro, and to correlate these with protein targets.

Microarray analyses were used to quantify activin A-induced changes in Sertoli cell miRNAs, and also mRNAs, after which miRNA-mediated target pathways were predicted by bioinformatic analysis. Of 411 Sertoli cell-expressed miRNAs, 60 miRNAs were >1.5-fold down-regulated while only one was >1.5-fold up-regulated, indicating that activin A suppresses most Sertoli cell miRNAs. Predicted pathway targets of these miRNAs and mRNAs included TGFβ-signalling and cell cycle. To confirm that activin-mediated miRNAs target genes in these pathways, two candidate genes (Smad 7, cyclin-D2) were chosen for further analysis. Smad-7, a known inhibitor of TGF-signalling, contains a target site for miRNA-322* in its 3’-UTR. The expression of both miRNA-322* and Smad 7 were regulated by activin A (1.5 fold down and 3.8 fold up, respectively). Similarly, the 3’-UTR of cyclin D2 contains target sites for miR-204* and miR-204, and all three molecules were regulated by activin (6 fold up, 2.6 fold down, 1.8 fold down, respectively).

We conclude that activin regulates a discrete subset of Sertoli cell miRNAs and genes, which target functions including TGFβ-signalling and cell proliferation, of likely importance in the de-differentiation of non-proliferative Sertoli cells to a functionally juvenile phenotype. These findings highlight potential new mechanisms of activin action in Sertoli cells in vivo.